Antibody Therapy

ABSTRACT

The present invention provides a composition comprising naked humanized, chimeric, and human anti-CEA antibodies and a therapeutic agent, which is useful for treatment of CEA expressing cancers and other diseases, and methods of use in treatment using this composition.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/639,298, filed Dec. 16, 2009, which was a divisional U.S. applicationSer. No. 11/932,530 (now U.S. Pat. No. 7,662,378), filed Oct. 31, 2007,which was a continuation of U.S. application Ser. No. 10/680,734 (nowU.S. Pat. No. 7,803,372), filed Oct. 8, 2003, which claimed the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Application No. 60/467,161,filed May 2, 2003. This application also claims priority toInternational Application No. PCT/US02/32307, filed Oct. 11, 2002, whichin turn claimed the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalApplication No. 60/416,531, filed Oct. 8, 2002.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The invention relates to methods of treating cancers that expresscarcinoembryonic antigen (“CEA”), particularly medullary thyroid cancer(MTC), non-medullary thyroid cancers (non-MTC), colorectal cancers,hepatocellular carcinoma, gastric cancer, lung cancer, breast cancer andother cancers, in which CEA is expressed, by administering animmunological reagent comprising an antibody in combination with atleast one other therapeutic agent, such as another antibody, achemotherapeutic agent, a radioactive agent, an antisenseoligonucleotide, an immunomodulator, an immunoconjugate or a combinationthereof. The invention further relates to pharmaceutical compositionscomprising the immunological reagent and at least one therapeutic agentin an unconjugated form. In particular, the invention relates to methodsof treating cancers that express CEA by administering, prior to, with orafter administering the therapeutic agent, a Class IIIanti-carcinoembryonic antigen (“anti-CEA”) monoclonal antibody (“MAb”),particularly a MAb, that has the binding affinity characteristics andspecificities of corresponding murine Class III anti-CEA MAb, and moreparticularly humanized, chimeric or human MAbs, that possess more of theantigenic and effector properties of a human antibody. Particularlyuseful MAbs in the method of treatment are humanized MAbs in which thecomplementarity-determining regions (“CDRs”) of an anti-CEA murine MAbare grafted into the framework regions of a human antibody.

B. Background

CEA is an oncofetal antigen commonly expressed in a number of epithelialcancers, most commonly those arising in the colon but also in thebreast, lung, pancreas, thyroid (medullary type) and ovary (Goldenberget al., J. Natl. Cancer Inst. 57:11-22 (1976), Shively, et al., Crit.Rev. Oncol. Hematol. 2:355-399 (1985)). CEA was originally thought to bea tumor-specific antigen of colorectal cancer (Gold et al., J. Exper.Med., 122:467 (1965)). However, it was later found to be present in adiverse number of carcinomas, benign tumors, and diseased tissues, aswell as in normal human colon (Shively et al., Crit. Rev. Oncol.Hematol., 2:355 (1985); von Kleist et al., Proc. Natl. Acad. Sci. USA.,69:2492 (1972)). CEA has been shown to mediate cell-cell adhesionthrough homotypic and heterotypic interactions, which in turn haveimplicated a role for CEA in various aspects of tumorigenesis.

Medullary thyroid cancer (MTC) confined to the thyroid gland ispotentially curable by total thyroidectomy and central lymph nodedissection. However, disease recurs in approximately 50% of thesepatients. In addition, the prognosis of patients with unresectabledisease or distant metastases is poor, less than 30% survive 10 years(Rossi et al., Amer. J. Surgery, 139:554 (1980); Samaan et al., J. Clin.Endocrinol. Metab., 67:801 (1988); Schroder et al., Cancer, 61:806(1988). These patients are left with few therapeutic choices (Principlesand Practice of Oncology, DeVita, Hellman and Rosenberg (eds.), NewYork: JB Lippincott Co. 1333-1435 (1989); Cance et al., Current ProblemsSurgery, 22:1 (1985)). Chemotherapy has been of little value andradiation therapy may only be used to control local disease (Cance etal.; Tubiana et al., Cancer, 55:2062 (1985)). Thus, new therapeuticmodalities are needed to control this disease.

A useful approach to cancer therapy and diagnosis involves the use oftargeting antibodies to deliver therapeutic and diagnostic agentsdirectly to the site of a malignancy. Over the past decade, a widevariety of tumor-specific antibodies and antibody fragments have beendeveloped, as have methods to conjugate the antibodies to therapeuticagents, such as drugs, toxins, radionuclides, immunomodulators, such ascytokines or other agents, and to administer the conjugates to patientsthat target the tumor. However, patients treated with drugs orradionuclides complexed with murine monoclonal antibodies (which havebeen the most commonly used targeting antibodies for humans) developcirculating human anti-mouse antibodies (HAMAs) and sometimes ageneralized immediate type-III hypersensitivity reaction to the antibodymoiety of the conjugate. But these problems have been minimized bymaking these murine antibodies less immunogenic by a number of differentmethods, which include making humanized, chimeric or human antibodies,by chemically modifying the targeting antibody, such as by conjugatingto polyethylene glycol to the targeting antibody (PEGylation), or bycharacterizing the situs of antigenicity in an antibody and thenremoving it; e.g., Fab′, F(ab)₂ and other antibody fragments have beenused in place of whole IgG. In addition, attempts have been made toreduce the adverse effects of HAMA by plasmaphoretically removing HAMAfrom blood. Immunosuppressive techniques also have been used toameliorate the adverse effect of the foreign antibody sufficiently topermit multiple treatments with the targeting agent.

Regardless of these treatment advances, there still exists a need toprovide more effective methods of treating CEA-expressing cancers. Thepresent invention provides an effective therapy utilizing anti-CEAantibodies, such as a Class III anti-CEA MAb, the murine MN-14 MAb asdefined in U.S. Pat. No. 5,874,540 and Hansen et al., Cancer, 71:3478(1993), and a Class III anti-CEA MAb, the chimeric and humanized MN-14MAbs as also defined in U.S. Pat. No. 5,874,540, and the NP-4 as definedin U.S. Pat. No. 4,818,709 by Primus et al., for example, allincorporated herein in their entirety by reference. Preferably, theClass III anti-CEA MAb is humanized, and used in combination with atherapeutic agent, particularly a chemotherapeutic agent, to yield aneffective therapeutic treatment for CEA expressing cancers with minimaltoxicity. Additionally, other anti-CEA antibodies, such Class II MAbs,for example, MN-6 (see Hansen et al., above, and NP-3 (se U.S. Pat. No.4,818,709), and Class I MAbs, such MN-3 and MN-15 (see also Hansen etal., above) provide effective methods of treating CEA expressingcancers. Further, the separate administration of these two componentsprovides enhanced results and the versatility and the flexibility totailor individual treatment methods.

SUMMARY OF THE INVENTION

Contemplated in the present invention are compositions and methods oftreating medullary and non-medullary thyroid carcinomas.

The first embodiment of the present invention is a compositioncomprising at least one anti-CEA monoclonal antibody (MAb) or fragmentthereof, which is preferably a Class III anti-CEA MAb or fragment, andat least one therapeutic agent. Preferably, the antibody fragment isselected from the group consisting of F(ab′)₂, Fab′, Fab, Fv and scFv.Also preferred, the Class III anti-CEA MAb or fragment thereof ishumanized, and wherein the humanized MAb retains substantially the ClassIII anti-CEA binding specificity of a murine Class III anti-CEA MAb.Also preferred, the Class III anti-CEA MAb or fragment thereof is achimeric MAb, and wherein the chimeric MAb retains substantially theClass III anti-CEA binding specificity of murine Class III anti-CEA MAb.Still preferred, the Class III anti-CEA MAb or fragment thereof is afully human MAb, and wherein said fully human MAb retains substantiallythe Class III anti-CEA binding specificity of murine Class III anti-CEAMAb. Other preferred anti-CEA Mabs for this purpose include Class IIMabs or fragments thereof, that are not CD66a-d cross-reactive which arediscussed in greater detail herein. Another embodiment includes Class IIanti-CEA Mabs or fragments thereof, that may react with CD66a, b and dbut not CD66c or Class I Mabs or fragments thereof, that react withCD66a, b, or d as well as CD66c (by definition a Class I Mab binds withCD66c).

In one embodiment of the present invention, the Class III anti-CEAmonoclonal antibody or fragment thereof is preferably a MN-14 antibodyor fragment thereof. More preferably, the MN-14 monoclonal antibody orfragment thereof comprises the complementarity-determining regions(CDRs) of a murine MN-14 monoclonal antibody, wherein the CDRs of thelight chain variable region of the MN-14 antibody comprises CDR1comprising the amino acid sequence KASQDVGTSVA (SEQ ID NO: 20); CDR2comprising the amino acid sequence WTSTRHT (SEQ ID NO: 21); and CDR3comprising the amino acid sequence QQYSLYRS (SEQ ID NO: 22); and theCDRs of the heavy chain variable region of the Class III anti-CEAantibody comprises CDR1 comprising TYWMS (SEQ ID NO: 23); CDR2comprising EIHPDSSTINYAPSLKD (SEQ ID NO: 24); and CDR3 comprisingLYFGFPWFAY (SEQ ID NO: 25). Also preferred, the MN-14 monoclonalantibody reacts with CEA and is unreactive with normal cross-reactiveantigen (NCA) and meconium antigen (MA). Most preferably, the MN-14monoclonal antibody or fragment thereof is a humanized, chimerized orfully human MN-14 antibody or fragment thereof.

In a preferred embodiment, the framework regions (FRs) of the light andheavy chain variable regions of the humanized MN-14 antibody or fragmentthereof comprise at least one amino acid substituted from thecorresponding FRs of a murine MN-14 monoclonal antibody. Specifically,the humanized MN-14 antibody or fragment thereof preferably comprises atleast one amino acid from the corresponding FR of the murine MN-14antibody is selected from the group consisting of amino acid residue 24(A), 28 (D), 30 (T), 48 (I), 49 (G), 74 (A) and 94 (S) of the murineheavy chain variable region (KLHuVhAIGA) of FIG. 14A-C. Likewise, thehumanized MN-14 antibody or fragment thereof may also comprise at leastone amino acid from said corresponding FR of the murine MN-14 lightchain variable region. Still preferred, the humanized MN-14 antibody orfragment thereof comprises the light chain variable region as set forthin FIG. 13A, and the heavy chain variable region set forth in FIG. 14A-Cdesignated as KLHuVhAIGA.

In the first embodiment of the present invention, the therapeutic agentis selected from the group consisting of a naked antibody, a cytotoxicagent, a drug, a radionuclide, an immunomodulator, a photoactivetherapeutic agent, an immunoconjugate, a hormone, or a combinationthereof, optionally formulated in a pharmaceutically acceptable vehicle.It is also contemplated herein that the therapeutic agent is notdacarbazine (DTIC).

The second embodiment of the present invention describes a method fortreating medullary as well as non-medullary thyroid carcinoma comprisingadministering to a subject, either concurrently or sequentially, atherapeutically effective amount a Class III anti-CEA monoclonalantibody or fragment thereof and at least one therapeutic agent, andoptionally formulated in a pharmaceutically acceptable vehicle.Preferably, the antibody fragment is selected from the group consistingof F(ab′)₂, Fab′, Fab, Fv and scFv. Also preferred, the Class IIIanti-CEA MAb or fragment thereof is humanized, wherein said humanizedMAb retains substantially the Class III anti-CEA binding specificity ofa murine Class III anti-CEA MAb. It is also contemplated that the ClassIII anti-CEA MAb or fragment thereof is a chimeric MAb, and wherein saidchimeric MAb retains substantially the Class III anti-CEA bindingspecificity of murine Class III anti-CEA MAb.

In a preferred embodiment, the Class III anti-CEA monoclonal antibody orfragment thereof is a MN-14 antibody or fragment thereof. Preferably,the MN-14 monoclonal antibody or fragment thereof comprises thecomplementarity-determining regions (CDRs) of a murine MN-14 monoclonalantibody, wherein the CDRs of the light chain variable region of saidMN-14 antibody comprises CDR1 comprising the amino acid sequenceKASQDVGTSVA (SEQ ID NO: 20); CDR2 comprising the amino acid sequenceWTSTRHT (SEQ ID NO: 21); and CDR3 comprising the amino acid sequenceQQYSLYRS (SEQ ID NO: 22); and the CDRs of the heavy chain variableregion of said Class III anti-CEA antibody comprises CDR1 comprisingTYWMS (SEQ ID NO: 23); CDR2 comprising EIHPDSSTINYAPSLKD (SEQ ID NO:24); and CDR3 comprising LYFGFPWFAY (SEQ ID NO: 25). Also preferred, theMN-14 monoclonal antibody is humanized, chimerized or fully human, andreacts with CEA and is unreactive with normal cross-reactive antigen(NCA) and meconium antigen. Also preferred, the MN-14 antibody orfragment thereof is administered in a dosage of 100 to 600 milligramsprotein per dose per injection. Most preferably, the MN-14 antibody orfragment thereof is administered in a dosage of 300-400 milligramsprotein per dose per injection.

In the methods of the instant invention, the framework regions (FRs) ofthe light and heavy chain variable regions of said humanized MN-14antibody or fragment thereof preferably comprise at least one amino acidsubstituted from the corresponding FRs of a murine MN-14 monoclonalantibody. More preferred, the humanized MN-14 antibody or fragmentthereof comprising at least one amino acid from said corresponding FR ofsaid murine MN-14 antibody is selected from the group consisting ofamino acid residue 24, 28, 30, 48, 49, 74 and 94 of the murine heavychain variable region of FIG. 14A-C, as noted above. Also preferred, thehumanized MN-14 antibody or fragment thereof comprising at least oneamino acid from said corresponding FR of said murine MN-14 light chainvariable region. Most preferably, the humanized MN-14 antibody orfragment thereof comprises the light chain variable region as set forthin FIG. 13A (middle sequence) or FIG. 22A (hMN-14) or FIG. 23A and theheavy chain variable region set forth in FIG. 14A-C designated asKLHuVhAIGA or FIG. 22B (hMn-14) or FIG. 23B.

The methods of the instant invention may further comprise administeringto a subject, either concurrently or sequentially, a therapeuticallyeffective amount of a second humanized, chimeric, human or murinemonoclonal antibody or fragment thereof selected from the groupconsisting of a monoclonal antibody or fragment thereof reactive withEGP-1, EGP-2 (e.g., 17-1A), IL-6, insulin-like growth factor-1, MUC-1,MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3,A33, Ep-CAM, AFP, Tn, Thomson-Friedenreich antigens, tumor necrosisantigens, VEGF, placenta growth factor (P1GF) or other tumorangiogenesis antigens, Ga 733, tenascin, fibronectin and a combinationthereof. Similarly, the methods may comprise administering to a subject,either concurrently or sequentially, a therapeutically effective amountof a second humanized, chimeric, human or murine monoclonal antibody orfragment thereof selected from the group consisting of a Class I orClass II or Class III anti-CEA monoclonal antibody or fragment thereofas described above. Preferably, the second antibody or fragment thereofis either naked or conjugated to a therapeutic agent.

In a preferred embodiment of the methods described herein, thetherapeutic agent is selected from the group consisting of a nakedantibody, cytotoxic agent, a drug, a radionuclide, an immunomodulator, aphotoactive therapeutic agent, an immunoconjugate of a CEA or non-CEAantibody, a hormone, or a combination thereof, optionally formulated ina pharmaceutically acceptable vehicle. It is also contemplated that thetherapeutic agent is not dacarbazine (DTIC).

Preferably, the therapeutic agent is a cytotoxic agent selected from thegroup consisting of a drug or a toxin. For example, it is contemplatedthat the drug possesses the pharmaceutical property selected from thegroup consisting of antimitotic, alkylating, antimetabolite,antiangiogenic, apoptotic, alkaloid, COX-2, and antibiotic agents andcombinations thereof. Preferably, the drug is selected from the groupconsisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,antimetabolites, antibiotics, enzymes, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, antagonists,endostatin, taxols, camptothecins, oxaliplatin, doxorubicins and theiranalogs, and a combination thereof.

When the therapeutic agent is a microbial, plant or animal toxin, theagent can be selected from the group consisting of ricin, abrin, alphatoxin, saporin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

It is also contemplated in the methods of the instant invention that thetherapeutic agent is an immunomodulator is selected from the groupconsisting of a cytokine, a stem cell growth factor, a lymphotoxin, ahematopoietic factor, a colony stimulating factor (CSF), an interferon(IFN), a stem cell growth factor, erythropoietin, thrombopoietin and acombination thereof. Preferably, the lymphotoxin is tumor necrosisfactor (TNF), said hematopoietic factor is an interleukin (IL), saidcolony stimulating factor is granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF)),said interferon is interferons-α, -β or -γ, and said stem cell growthfactor is designated “S1 factor”. Also preferred, the immunomodulatorcomprises IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21,interferon-γ, TNF-α or a combination thereof. Administration of acytokine prior to, simultaneous with, or subsequent to exposure to acytotoxic agent that results in myeloid or hematopoietic toxicity isdescribed in U.S. Pat. No. 5,120,525, which is incorporated herein byreference in its entirety.

Also preferred the therapeutic agent is a photoactive therapeutic agentthat is a chromogen or dye or an alkylating agent that is dacarbazine.

Also preferred, the therapeutic agent is a radionuclide that has anenergy between 20 and 10,000 keV. Preferably, the radionuclide isselected from the group consisting of ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁸Y, ²²⁵Ac,¹⁷⁷Lu, ¹⁸⁸Re, ¹⁸⁶Re, and combinations thereof.

In another embodiment, an immunomodulator, as described herein, isadministered prior to the administration of a therapeutically effectiveamount of a anti-CEA monoclonal antibody or fragment thereof alone or animmunomodulator is administered prior to the administration of atherapeutically effective amount of a anti-CEA monoclonal antibody andat least one therapeutic agent, wherein any of these componentsdescribed herein are optionally formulated in a pharmaceuticallyacceptable vehicle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Graphs comparing tumor volume after treatment with hMN-14 alone,DTIC alone or the combination of hMN-14 and DTIC. FIG. 1A shows DTICadministered alone at 25 and 100 μg/dose or with 250 μg hMN-14 antibodyand FIG. 1B shows DTIC administered alone at 50 and 75 μg/dose or with100 μg hMN-14 antibody.

FIG. 2. Graph comparing tumor volume after radioimmunotherapy (RAIT)with ¹³¹I and ⁹⁰Y-MN-14.

FIG. 3. Graph comparing the therapeutic efficacy of severalchemotherapeutic drugs on tumor volume in TT bearing mice. TT bearingmice were given doxorubicin @ 20 mg/m²; days 0, 1, and 2, (70 μg/dose)(O); DTIC @ 300 mg/m²; days 0, 1, and 2, (1.08 mg/dose) (□); doxorubicinand DTIC as above (); cyclophosphamide @ 600 mg/m²; day 0 (2.16mg/dose) (Δ); vincristine @ 4.2 μg/dose; day 0 (x); all 4 drugs(doxorubicin, DTIC, cyclophosphamide, and vincristine) at the dosesdescribed for each above (▪); or left untreated (♦). Groups consisted of6-9 nude mice bearing established TT tumors. Mean tumor volume at timeof treatment was 0.51+/−0.33 cm³. Points: mean tumor size. Error bars:std dev, and are shown only above the symbol for clarity.

FIG. 4. Graph comparing the therapeutic efficacy of combination therapyof RAIT with ⁹⁰Y-labeled anti CEA MAb MN-14 and a 4-drug combinationinitiated 24 hours after RAIT on tumor volume in mice. Tumor bearinganimals were either left untreated (♦); given the 4-drug regimendescribed in FIG. 1, administered on days 1, 2, and 3 (▪); 52.5 μCi⁹⁰Y-MN-14 day on 0, followed by the 4-drug regimen administered on days1, 2, and 3 (▴); 52.5 μCi ⁹⁰Y-MN-14 on day 0 (Δ); or 105 μCi ⁹⁰Y-MN-14on day 0 (∘). N=5 for the untreated group, and n=9-10 in the treatmentgroups. Mean tumor volume at time of treatment was 0.28+/−0.12 cm³.Points: mean tumor size. Error bars: std dev, and are shown only abovethe symbol for clarity.

FIG. 5. Graph comparing the efficacy of RAIT plus DTIC and RAIT plusdoxorubicin and DTIC in TT bearing mice. TT bearing mice were eitherleft untreated (♦); given 105 μCi ⁹⁰Y-MN-14 on day 0 (∘); 105 μCi⁹⁰Y-MN-14 on day 0, followed by the doxorubicin and DTIC regimenadministered at 50% the full dosage on days 1, 2, and 3 (▴); 105 μCi⁹⁰Y-MN-14 day on 0, followed by DTIC at 75% of the full dosage on days1, 2, and 3 (x); or the full dosage of DTIC, 300 mg/m² on days 1, 2, and3, (1.08 mg/dose) (□). N=5 for the untreated group, and n=8-9 in thetreatment groups. Mean tumor volume at time of treatment was 0.39+/−0.20cm³. Points: mean tumor size. Error bars: std dev, and are shown onlyabove the symbol for clarity.

FIG. 6. Graph comparing the effectiveness of naked hMN-14 with treatmentregimens in mice bearing TT xenografts. Animals were given s.c.injections of TT cells, and either left untreated (A) or given an i.v.injection of 0.5 mg hMN-14 1 day (B) or 11 days (C) later. Lines inpanels A, B, and C represent tumor volumes of individual animals. Meansof respective treatment groups are shown in panel D. Error barsrepresent standard error of the mean and are shown only in one directionfor clarity. ♦, untreated; □, day-1 treated; Δ, day-11 treated.

FIG. 7. Graph comparing the effectiveness of humanized and murine MN-14antibodies in treating medullary thyroid carcinoma. Animals were givens.c. injections of TT cells, and either left untreated or given an i.v.injection of MAb (0.5 mg) 1 day later. Means of respective treatmentgroups are shown. Error bars represent standard error of the mean andare shown only in one direction for clarity. ♦, untreated; □, hMN-14; Δ,murine MN-14; x, hLL2.

FIG. 8. Graph comparing the effectiveness of different hMN-14 doses intreating medullary thyroid carcinoma. Animals were given i.v. injectionsof increasing doses of hMN-14 1 day after s.c. injection of TT cells.Means of respective treatment groups are shown. ♦, untreated; , 0.125mg; ∘, 0.25 mg; ▪, 0.50 mg; x, 1.0 mg, ▴, 2.0 mg. Error bars representstandard error of the mean and are shown only for the untreated groupand the group that received 0.50 mg hMN-14/mouse for clarity.

FIG. 9. Graph comparing the effectiveness of different treatment timesin TT bearing nude mice. Animals were given i.v. injections of 0.25hMN-14 either 1 (), 3 (▴), or 7 (▪) days after s.c. injection of TTcells, or left untreated (♦). Means of respective treatment groups areshown. Error bars represent standard error of the mean and are shownonly in one direction for clarity.

FIG. 10. Graph comparing treatment of TT bearing nude mice with hMN-14plus DTIC, DTIC alone, hMN-14 alone, and untreated mice. Animals weregiven i.p. injections of hMN-14 at 100 μg/dose on days 2, 3, 4, 5, 7, 8,9, 10 and 11, 15, and 22, then every 7 days (∘); DTIC, 75 μg/dose ondays 2, 3, and 4 (▴); the combination of these hMN-14 and DTIC regimens(Δ); or left untreated (♦). Means of respective treatment groups areshown; 10 animals/group.

FIG. 11. FIGS. 11A and 11B show the consensus DNA sequence of the murineMN-14 variable region heavy chain (VH) (SEQ ID NO: 1) and the amino acidsequence (SEQ ID NO: 2) encoded by the DNA sequence. The CDRs areenclosed in boxes.

FIG. 12. FIGS. 12A and 12B show the consensus DNA sequence of the murineMN-14 variable region light chain (VK) (SEQ ID NO: 3) and the amino acidsequence (SEQ ID NO: 4) encoded by the DNA sequence. The CDRs areenclosed in boxes.

FIG. 13. FIGS. 13A and 13B show the alignment of the murine MN-14variable region (MN14VH is shown in SEQ ID NO: 2, MN14VK is shown in SEQID NO: 4) of the with the human variable regions NEWM VH (SEQ ID NO: 5)and REI VK (SEQ ID NO: 6) (FIG. 13A), and with the human KOL VH region(SEQ ID NO: 7) (FIG. 13B). CDRs are boxed, and the murine VH FRs, whichare incorporated into the humanized VH, are marked with their positionsaccording to the numbering system of Kabat et al. SEQUENCES OF PROTEINSOF IMMUNOLOGICAL INTEREST, U.S. Government Printing Office, Washington,D.C., 1987. Murine residues outside the CDRs that were included in theKLHuVH are indicated by a filled circle.

FIG. 14. FIGS. 14A-14C show a comparison of the amino acid sequencebetween murine and humanized MN-14 VH framework residues (FR) (SEQ IDNOS 2, 26, 8-11, 27 and 12-15, respectively in order of appearance).Only human FR residues different from the mouse are shown. CDRs for NEWMand KOL are also not shown. The areas of amino acid substitutions in therespective FRs are highlighted in bold, and the position of thesubstitution indicated according to the Kabat et al. numbering system.The 3 CDRs are boxed.

FIG. 15. FIG. 15 shows the effects of naked hMN-14 CEA MAb and DTICtreatment in a human medullary thyroid cancer model.

FIG. 16. FIG. 16 shows the effects of naked hMN-14 CEA MAb and CPT-11treatment in an advanced human colon cancer model.

FIG. 17. FIG. 17 shows the effects of naked hMN-14 CEA MAb and CPT-11treatment in a low tumor burden human colon cancer model.

FIG. 18. FIG. 18 shows the effects of pre-treatment with naked hMN-14CEA MAb given 3 days prior to CPT-11 treatment in a human colon cancermodel.

FIG. 19. FIG. 19 shows a comparison of various administration sequencesof naked hMN-14 CEA Mab and CPT-11 in a human colon cancer model.

FIG. 20. FIG. 20 shows the effects of GM-CSF pre-treatment on nakedhMN-14 CEA MAb therapy in a human colon cancer model.

FIG. 21. FIG. 21 shows a comparison of the effects of naked hMN-14 CEAMAb therapy on both low CEA expression and high (interferon-induced) CEAexpression tumor cells in a human colon cancer model.

FIG. 22. FIGS. 22A and 22B show the comparison of the human, murine andhumanized sequences of the Vk and VH regions of the human REI and KOLantibodies, respectively with murine and humanized MN-14. The humansequences of the REI Vk (SEQ ID NO: 6) in FIG. 22A are compared with themurine (SEQ ID NO: 4) and humanized (SEQ ID NO: 19) MN-14 Vk sequences.The closed circles indicate sequences retained from the human REI Vksequences. The CDRs are boxed. The human sequences of the KOL VH (SEQ IDNO: 7) in FIG. 22B are compared with the murine (SEQ ID NO: 2) andhumanized (SEQ ID NO: 14) MN-14 VH sequences. The closed circlesindicate sequences retained from the human KOL VH sequences. The CDRsare boxed.

FIG. 23. FIGS. 23A and 23B show the Vk, the variable light chain, andthe VH, the variable heavy chain sequences of hMN-14, a humanized ClassIII anti-CEA antibody. The CDR region sequences are shown in bold andunderlined. The amino acid residues and the nucleotides are numberedsequentially. The light chain variable region is shown in FIG. 23A(Nucleotide and encoded protein are disclosed as SEQ ID NOS 18 and 19,respectively), and the heavy chain variable region is shown in FIG. 23B(Nucleotide and encoded protein are disclosed as SEQ ID NOS 16 and 17,respectively).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Overview

The present invention provides methods of treatment in which a nakedanti-CEA antibody or fragment thereof and at least one therapeutic agentare administered either sequentially or concurrently over a treatmentperiod. The method is particularly useful for treating medullary thyroidcarcinoma but is surprisingly useful for treating non-medullary thyroidcancers, colorectal cancers, hepatocellular carcinoma, pancreatic,breast, lung, head-and-neck, bladder, uterine and ovarian cancers, andeven cancers that do not express CEA at very high levels. For example,treatment is contemplated in cancers that express CEA at levels of atleast 100 ng/g of tissue. The present method further providescompositions comprising the anti-CEA antibody, which is preferably aClass III anti-CEA antibody or antibody fragment in which the antibodyand the therapeutic agent are not conjugated or linked to each other. Asused herein, the phrase “Class III anti-CEA” antibody or antibodyfragment means an antibody or fragment that binds the CEA antigen (orCD66e) and is unreactive with normal cross-reactive antigen (NCA),meconium antigen (MA), granulocytes and CD66a-d (see, Primus et al.,U.S. Pat. No. 4,818,709, incorporated by reference). The naked Class IIIanti-CEA antibody or fragment thereof may be a humanized, chimeric,human or murine antibody. In a preferred embodiment, the naked Class IIIanti-CEA antibody or fragment thereof is a humanized MN-14 antibody orfragment thereof.

Also contemplated for use in the present invention are Class II Mabsthat are not CD66a-d cross-reactive. These are Mabs that are reactivewith CEA domains N-A1B1, A2B2, which do react with Meconium Antigen, butnot with NCA, and do not react with granulocytes. For example, NP-3 andMN-6 are Class II anti-CEA antibodies useful in the present invention.Additionally contemplated for use in the present invention are Class IIanti-CEA Mabs or fragments thereof, that may react with CD66a, b and dbut not CD66c or Class I Mabs or fragments thereof, that react withCD66a, b, or d as well as CD66c (by definition a Class I Mab binds withCD66c).

Surprisingly, the compositions and methods described herein are alsouseful for treating non-medullary thyroid carcinoma, includingcolorectal cancer, pancreatic cancer, breast cancer, lung cancers,hepatocellular carcinoma, urinary bladder cancer, head-and-neck cancers,and ovarian cancer. Because such forms of cancer express less CEA thanmedullary thyroid cancers, it was unexpected that a naked Class IIIanti-CEA antibody, in combination with a therapeutic agent, would beuseful for treating non-medullary thyroid carcinomas.

The mechanism of tumor cell killing by the naked Class III anti-CEAantibody is not known with certainty and is likely involves severalmechanisms. It is hypothesized that the naked antibody alone or incombination with the therapeutic agent may affect tumor growth byblocking biological activities of their respective antigens or bystimulating natural immunological functions, such as antibody-dependentcell-mediated cytotoxicity (ADCC) or complement-mediated lysis.Additionally, the naked antibody alone or in combination with thetherapeutic agent may treat and control the cancer by inhibiting cellgrowth and cell cycle progression, inducing apoptosis, inhibitingangiogenesis, inhibiting metastatic activity, and/or affecting tumorcell adhesion. In fact, the anti-CEA antibody or fragment thereof of thepresent invention may be more effective in treating metastases thanprimary cancers, since the metastases may be more susceptible toantagonists of tumor cell adhesion. The present treatment methodprovides a treatment plan that may be optimized to provide the maximumanti-tumor activity for individual patients by allowing the titration ofthe antibody and one or more different therapeutic agents to provide aneffective treatment regimen.

In one aspect of the present invention, the naked Class III anti-CEAantibody or fragment thereof and therapeutic agent may be supplementedwith at least one additional therapeutic agent, such as a naked orconjugated humanized, murine, chimeric or human antibody, fusionprotein, or fragment thereof. For example, another class III CEAantibody or antibody fragment that is non-blocking and does not bindgranulocytes or CD66a-d; a Class II anti-CEA antibody or antibodyfragment that is non-blocking and does not bind granulocytes or CD66a-d;a Class II anti-CEA Mabs or fragments thereof, that may react withCD66a, b and d but not CD66c, Class I Mabs or fragments thereof, thatreact with CD66a, b, or d as well as CD66c (by definition a Class I Mabbinds with CD66c) or an antibody against a differentcarcinoma-associated epitope or antigen, may be used as the therapeuticagent for combination therapy with the preferred humanized MN-14antibody. Such an additional antibody, fusion protein or fragmentthereof may bind CEA or another cancer or tumor-associated antigen, asdescribed in more detail below.

2. Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv) and the like. Regardlessof structure, an antibody fragment binds with the same antigen that isrecognized by the intact antibody.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the variable regions, such as the “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region. The Fv fragments may beconstructed in different ways as to yield multivalent and/ormultispecific binding forms. In the former case of multivalent, theyreact with more than one binding site against the CEA epitope, whereaswith multispecific forms, more than one epitope (either of CEA or evenagainst CEA and a different antigen) is bound.

As used herein, the term antibody component includes both an entireantibody, a fusion protein, and fragments thereof.

A naked antibody is generally an entire antibody which is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector or immunological functions, such ascomplement fixation and ADCC (antibody dependent cell cytotoxicity),which set mechanisms into action that may result in cell lysis. However,the Fc portion may not be required for therapeutic function of theantibody, but rather other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,such as inhibition of heterotypic or homotypic adhesion, andinterference in signaling pathways, may come into play and interferewith the disease progression. Naked antibodies include both polyclonaland monoclonal antibodies, and fragments thereof, that include murineantibodies, as well as certain recombinant antibodies, such as chimeric,humanized or human antibodies and fragments thereof. As defined in thepresent invention, “naked” is synonymous with “unconjugated,” and meansnot linked or conjugated to the therapeutic agent with which itadministered.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, while the constant domains ofthe antibody molecule are derived from those of a human antibody. Forveterinary applications, the constant domains of the chimeric antibodymay be derived from that of other species, such as a cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule is derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody component,i.e., an antibody or antibody fragment, or a subfragment thereof, and isuseful in the treatment of a disease. Examples of therapeutic agentsinclude antibodies, antibody fragments, immunoconjugates, drugs,cytotoxic agents, toxins, nucleases, hormones, immunomodulators,chelators, boron compounds, photoactive agents or dyes, radioisotopes orradionuclides, antisense oligonucleotides, immunoconjugates orcombinations thereof.

An immunoconjugate is an antibody component conjugated to a therapeuticagent. Suitable therapeutic agents are described above.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which two or more ofthe same or different natural antibody, single-chain antibody orantibody fragment segments with the same or different specificities arelinked. A Class III anti-CEA fusion protein comprises at least one CEAbinding site. Preferably, the Class III anti-CEA fusion protein is aMN-14 fusion protein.

Valency of the fusion protein indicates the total number of binding armsor sites the fusion protein has to antigen(s) or epitope(s); i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody fusion protein means that it can take advantage of multipleinteractions in binding to an antigen, thus increasing the avidity ofbinding to the antigen, or to different antigens. Specificity indicateshow many different types of antigen or epitope an antibody fusionprotein is able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one type of antigen or epitope. A monospecific,multivalent fusion protein has more than one binding site for the sameantigen or epitope. For example, a monospecific diabody is a fusionprotein with two binding sites reactive with the same antigen. Thefusion protein may comprise a multivalent or multispecific combinationof different antibody components or multiple copies of the same antibodycomponent. For example, the fusion protein of the present invention maybe multispecific, wherein one arm of the fusion protein (e.g., scFv orFab) is a Class III, anti-CEA mAb that targets CD66e and another arm ofthe fusion protein is from another CEA crossreactive antibody thattargets CD66a-d.

A preferred bispecific fusion protein according to the invention has anarm against a Class III CEA epitope, and a second arm against CD66a-depitopes (Class II) expressed on granulocytes. In these embodiments, theCD66a-d binding portion should not be able to fix complement or bind toFc-receptors to effect ADCC (which would result in release of cytokinesfrom granulocytes). Though complement fixation and effecting ADCC arepreferred properties for the naked therapy embodiments of the presentinvention, they should be avoided in the context of the instantembodiments relating to bispecific fusion proteins. On normal coloncells NCA-50/90 and CEA are both expressed, but they are restricted tothe apical face of the normal epithelial cell, and this face ispresented only to the colon lumen, and not accessible to injectedantibody. CEA released from these normal cells as CEA, or bound to deadnormal cells is eliminated in the feces. This polarization is lost whena colon cancer develops, and both NCA-50/90 and CEA are then expressedon the cancer cell membrane that is invading the underlying normalbasement membrane which anchors the normal epithelial cells. Abispecific antibody such as hMN3/hMN14 is expected to react with bothCEA and NCA-50/90 on these invading cells. Furthermore, as NCA50/90 ispresent on granulocytes this bispecific is expected to directgranulocytes to kill the invading colon cancer cells. An even morepreferred construct according to this embodiment is a bispecific,trivalent protein with one arm reactive with NCA50/90 and two armsreactive with only CEA. Another embodiment would be a bispecific proteinwith two arms that bind to NCA50/90.

A preferred fusion protein also reactive with granulocytes would be adiabody, having one arm against NCA-50/90 (example hMN-3), and one armagainst a Class III epitope on CEA (hMN14). These fusion proteins do nothave an Fc-domain so they will not activate cytokine release fromgranulocytes. An even more preferred fusion protein would be a triabodywith one hMN-3 arm and two hMN14 arms. The construction of suchdiabodies and triabodies is disclosed in U.S. application Ser. Nos.60/404,919 (filed Aug. 22, 2002), 60/345,641 (filed Jan. 8, 2002),60/328,835 (filed Oct. 15, 2001), and 60/341,881 (filed Dec. 21, 2001).

Any kind of multispecific antibody made with mabs of the hMN14/NP-3specificities are also preferred and can have an Fc-domain able to fixcomplement/activate ADCC. For example, a hMN14-IgG1/[NP-3-scFv]2 fusionprotein could be used; the making of which is taught in U.S. applicationSer. No. 09/337,756.

Yet another preferred type of multispecific antibody according to thepresent invention is an hMN-3 MAb which has an Fc-domain lacking theability to fix complement/effect-ADCC.

The fusion protein may additionally comprise a therapeutic agent. Forexample, where at least one of the antibodies or fragments thereof, suchas the Class III, anti-CEA mAb that targets CD66e or its scFv or Fab maybe conjugated to cytokines, such as interferon or a colony-stimulatingfactor, such as GM-CSF or G-CSF or an interleukin, all of which aredescribed herein.

An immunomodulator is a therapeutic agent as defined in the presentinvention that when present, alters, suppresses or stimulates the body'simmune system. Typically, the immunomodulator useful in the presentinvention stimulates immune cells to proliferate or become activated inan immune response cascade, such as macrophages, B-cells, and/orT-cells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat are released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which act as intercellular mediatorsbetween cells. As the skilled artisan will understand, examples ofcytokines include lymphokines, monokines, interleukins, and severalrelated signalling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation.

Preparation of Monoclonal Antibodies, Including Chimeric, Humanized andHuman Antibodies

Monoclonal antibodies (MAbs) are a homogeneous population of antibodiesto a particular antigen and the antibody comprises only one type ofantigen binding site and binds to only one epitope on an antigenicdeterminant. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art. See, for example,Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.),CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies canbe obtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Abs to peptide backbones are generated by well-known methods for Abproduction. For example, injection of an immunogen, such as(peptide)_(n)-KLH, wherein KLH is keyhole limpet hemocyanin, and n=1-30,in complete Freund's adjuvant, followed by two subsequent injections ofthe same immunogen suspended in incomplete Freund's adjuvant intoimmunocompetent animals. The animals are given a final i.v. boost ofantigen, followed by spleen cell harvesting three days later. Harvestedspleen cells are then fused with Sp2/0-Ag14 myeloma cells and culturesupernatants of the resulting clones analyzed for anti-peptidereactivity using a direct-binding ELISA. Fine specificity of generatedAbs can be analyzed for by using peptide fragments of the originalimmunogen. These fragments can be prepared readily using an automatedpeptide synthesizer. For Ab production, enzyme-deficient hybridomas areisolated to enable selection of fused cell lines. This technique alsocan be used to raise antibodies to one or more of the chelatescomprising the linker, e.g., In(III)-DTPA chelates. Monoclonal mouseantibodies to an In(III)-di-DTPA are known (U.S. Pat. No. 5,256,395 toBarbet).

Another method for producing antibodies is by production in the milk oftransgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63:141-147, 1998; U.S. Pat. No. 5,827,690, both of which are incorporatedin their entirety by reference. Two DNA constructs are prepared whichcontain, respectively, DNA segments encoding paired immunoglobulin heavyand light chains. The DNA segments are cloned into expression vectorsthat contain a promoter sequence that is preferentially expressed inmammary epithelial cells. Examples include, but are not limited to,promoters from rabbit, cow and sheep casein genes, the cowα-lactoglobulin gene, the sheep β-lactoglobulin gene and the mouse wheyacid protein gene. Preferably, the inserted fragment is flanked on its3′ side by cognate genomic sequences from a mammary-specific gene. Thisprovides a polyadenylation site and transcript-stabilizing sequences.The expression cassettes are coinjected into the pronuclei offertilized, mammalian eggs, which are then implanted into the uterus ofa recipient female and allowed to gestate. After birth, the progeny arescreened for the presence of both transgenes by Southern analysis. Inorder for the antibody to be present, both heavy and light chain genesmust be expressed concurrently in the same cell. Milk from transgenicfemales is analyzed for the presence and functionality of the antibodyor antibody fragment using standard immunological methods known in theart. The antibody can be purified from the milk using standard methodsknown in the art.

After the initial raising of antibodies to the immunogen, the variablegenes of the monoclonal antibodies can be cloned from the hybridomacells, sequenced and subsequently prepared by recombinant techniques.General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Nat'l Acad: Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety. Humanization and chimerization of murine antibodies andantibody fragments are well known to those skilled in the art. Achimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. The use of antibodycomponents derived from humanized and chimerized monoclonal antibodiesalleviates potential problems associated with the immunogenicity ofmurine constant regions. Techniques for constructing chimeric antibodiesare well known to those of skill in the art. As an example, Leung etal., Hybridoma 13:469 (1994), describe how they produced an LL2 chimeraby combining DNA sequences encoding the V_(k) and V_(H) domains of LL2monoclonal antibody, an anti-CD22 antibody, with respective human K andIgG₁ constant region domains.

A chimeric monoclonal antibody (MAb) can also be humanized by replacingthe sequences of the murine FR in the variable domains of the chimericMAb with one or more different human FR. Specifically, humanizedmonoclonal antibodies are produced by transferring mouse complementarydetermining regions from heavy and light variable chains of the mouseimmunoglobulin into a human variable domain, and then, substitutinghuman residues in the framework regions of the murine counterparts. Assimply transferring mouse CDRs into human FRs often results in areduction or even loss of antibody affinity, additional modificationmight be required in order to restore the original affinity of themurine antibody. This can be accomplished by the replacement of one ormore human residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988).

In a preferred embodiment, some human residues in the framework regionsof the humanized anti-CEA antibody or fragments thereof are replaced bytheir murine counterparts. Additionally, knowing that chimeric anti-CEAexhibits a binding affinity comparable to that of its murinecounterpart, defective designs, if any, in the original version of thehumanized anti-CEA MAb can be identified by mixing and matching thelight and heavy chains of the chimeric anti-CEA to those of thehumanized version. Preferably, the humanized anti-CEA antibody is ahumanized MN-14 antibody, and it preparation and sequences are disclosedin U.S. Pat. No. 5,874,540, which is incorporated in its entirety byreference. Although the two human antibodies are REI and NEWM are thepreferred antibodies for preparing both humanized and chimeric MN-14antibodies, a combination of framework sequences from 2 or moredifferent human antibodies can be used for V_(H) and V_(K). Theproduction of humanized MAbs are described, for example, by Jones etal., Nature 321: 522 (1986), Riechmann et al., Nature 332:323 (1988),Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437(1992), and Singer et al., J. Immun. 150: 2844 (1993), each of which ishereby incorporated by reference. Further, the affinity of humanized,chimeric and human MAbs to a specific epitope can be increased bymutagenesis of the CDRs, so that a lower dose of antibody may be aseffective as a higher dose of a lower affinity MAb prior to mutagenesis.See for example, WO0029584A1.

In another embodiment, an antibody of the present invention is a humanClass III anti-CEA monoclonal antibody. The anti-CEA MAb, or anotherhuman antibody, can be obtained from a transgenic non-human animal. See,e.g., Mendez et al., Nature Genetics, 15: 146-156 (1997) and U.S. Pat.No. 5,633,425, which are incorporated in their entirety by reference.For example, a human antibody can be recovered from a transgenic mousepossessing human immunoglobulin loci. Preferably, the anti-CEA antibodyis an MN-14 antibody. The mouse humoral immune system is humanized byinactivating the endogenous immunoglobulin genes and introducing humanimmunoglobulin loci. The human immunoglobulin loci are exceedinglycomplex and comprise a large number of discrete segments which togetheroccupy almost 0.2% of the human genome. To ensure that transgenic miceare capable of producing adequate repertoires of antibodies, largeportions of human heavy- and light-chain loci must be introduced intothe mouse genome. This is accomplished in a stepwise process beginningwith the formation of yeast artificial chromosomes (YACs) containingeither human heavy- or light-chain immunoglobulin loci in germlineconfiguration. Since each insert is approximately 1 Mb in size, YACconstruction requires homologous recombination of overlapping fragmentsof the immunoglobulin loci. The two YACs, one containing the heavy-chainloci and one containing the light-chain loci, are introduced separatelyinto mice via fusion of YAC-containing yeast spheroblasts with mouseembryonic stem cells. Embryonic stem cell clones are then microinjectedinto mouse blastocysts. Resulting chimeric males are screened for theirability to transmit the YAC through their germline and are bred withmice deficient in murine antibody production. Breeding the twotransgenic strains, one containing the human heavy-chain loci and theother containing the human light-chain loci, creates progeny whichproduce human antibodies in response to immunization.

Unrearranged human immunoglobulin genes also can be introduced intomouse embryonic stem cells via microcell-mediated chromosome transfer(MMCT). See, e.g., Tomizuka et al., Nature Genetics, 16: 133 (1997). Inthis methodology microcells containing human chromosomes are fused withmouse embryonic stem cells. Transferred chromosomes are stably retained,and adult chimeras exhibit proper tissue-specific expression.

As an alternative, an antibody or antibody fragment of the presentinvention may be derived from human antibody fragments isolated from acombinatorial immunoglobulin library. See, e.g., Barbas et al., METHODS:A Companion to Methods in Enzymology 2:119 (1991), and Winter et al.,Ann. Rev. Immunol. 12: 433 (1994), which are incorporated by reference.Many of the difficulties associated with generating monoclonalantibodies by B-cell immortalization can be overcome by engineering andexpressing antibody fragments in E. coli, using phage display. To ensurethe recovery of high affinity, monoclonal antibodies a combinatorialimmunoglobulin library must contain a large repertoire size. A typicalstrategy utilizes mRNA obtained from lymphocytes or spleen cells ofimmunized mice to synthesize cDNA using reverse transcriptase. Theheavy- and light-chain genes are amplified separately by PCR and ligatedinto phage cloning vectors. Two different libraries are produced, onecontaining the heavy-chain genes and one containing the light-chaingenes. Phage DNA is isolated from each library, and the heavy- andlight-chain sequences are ligated together and packaged to form acombinatorial library. Each phage contains a random pair of heavy- andlight-chain cDNAs and upon infection of E. coli directs the expressionof the antibody chains in infected cells. To identify an antibody thatrecognizes the antigen of interest, the phage library is plated, and theantibody molecules present in the plaques are transferred to filters.The filters are incubated with radioactively labeled antigen and thenwashed to remove excess unbound ligand. A radioactive spot on theautoradiogram identifies a plaque that contains an antibody that bindsthe antigen. Cloning and expression vectors that are useful forproducing a human immunoglobulin phage library can be obtained, forexample, from STRATAGENE Cloning Systems (La Jolla, Calif.).

In one embodiment, the antibodies of the present invention are producedas described in Hansen et al., U.S. Pat. No. 5,874,540; Hansen et al.,Cancer, 71:3478 (1993); Primus et al., U.S. Pat. No. 4,818,709, andShively et al., U.S. Pat. No. 5,081,235, which have been incorporated byreference in their entirety.

Production of Antibody Fragments

The present invention contemplates the use of fragments of a Class IIIanti-CEA antibody, preferably a MN-14 antibody. The Class III anti-CEAantibody or fragment thereof of the present invention does not bindgranulocytes or CD66a-d. Antibody fragments which recognize specificepitopes can be generated by known techniques. For example, antibodyfragments can be prepared by proteolytic hydrolysis of an antibody or byexpression in E. coli of the DNA coding for the fragment. The antibodyfragments are antigen binding portions of an antibody, such as F(ab′)₂,Fab′, Fab, Fv, scFv and the like, and can be obtained by pepsin orpapain digestion of whole antibodies by conventional methods.

For example, an antibody fragment can be produced by enzymatic cleavageof antibodies with pepsin to provide a 100 Kd fragment denoted F(ab′)₂.This fragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 50 Kd Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using papain producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein, which patents areincorporated herein in their entireties by reference. Also, see Nisonoffet al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73:119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described in Inbar etal., Proc. Nat'l. Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992).

Preferably, the Fv fragments comprise V_(H) and V_(L) chains which areconnected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the V_(H) and V_(L) domains which are connectedby an oligonucleotide. The structural gene is inserted into anexpression vector that is subsequently introduced into a host cell, suchas E. coli. The recombinant host cells synthesize a single polypeptidechain with a linker peptide bridging the two V domains. Methods forproducing sFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology, 2:97 (1991). Also see Bird et al.,Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778; Pack etal., Bio Technology 11:1271 (1993) and Sandhu, supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2:106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Humanized, Chimeric and Human Anti-CEA Antibodies for Treatment

Described in the present invention are compositions and methods usingmurine, chimeric, humanized and human Class III anti-CEA antibodies andfragments thereof for treatment. Preferably, the Class III anti-CEAantibody or fragment thereof is a MN-14 antibody or fragment thereof.The antibodies of the present invention can be used to treat medullarythyroid carcinoma (MTC), as well as non-MTC CEA-expressing carcinomas.Exemplary non-MTC CEA expressing carcinomas include colorectal cancer,pancreatic cancer, hepatocellular carcinoma, gastric cancer, lungcancer, head- and neck cancers, urinary bladder cancer, uterine cancer,breast cancer, and ovarian cancer.

Compositions

Contemplated herein is a composition comprising at least one Class IIIanti-CEA monoclonal antibody (MAb) or fragment thereof and at least onetherapeutic agent, which are not conjugated to each other, and thus arepresent in the composition as unconjugated forms of each of thecomponents. In compositions comprising more than one antibody orantibody fragments, such as a second Class III anti-CEA antibody, thesecond antibody is non-blocking (i.e., does not block binding of thefirst Class III anti-CEA antibody or antibody fragment).

In one embodiment, the Class III anti-CEA monoclonal antibody orfragment thereof is humanized, chimeric, or fully human, wherein thehumanized, chimeric, or fully human MAb retains substantially the ClassIII anti-CEA binding specificity of a murine Class III anti-CEA MAb.

In a preferred embodiment, the Class III anti-CEA monoclonal antibody orfragment thereof is a MN-14 antibody or fragment thereof. Preferably,the MN-14 monoclonal antibody or fragment thereof comprises thecomplementarity-determining regions (CDRs) of a murine MN-14 monoclonalantibody, wherein the CDRs of the light chain variable region of saidMN-14 antibody comprises CDR1 comprising the amino acid sequenceKASQDVGTSVA (SEQ ID NO: 20); CDR2 comprising the amino acid sequenceWTSTRHT (SEQ ID NO: 21); and CDR3 comprising the amino acid sequenceQQYSLYRS (SEQ ID NO: 22); and the CDRs of the heavy chain variableregion of said Class III anti-CEA antibody comprises CDR1 comprisingTYWMS (SEQ ID NO: 23); CDR2 comprising EIHPDSSTINYAPSLKD (SEQ ID NO:24); and CDR3 comprising LYFGFPWFAY (SEQ ID NO: 25). Also preferred, theMN-14 monoclonal antibody reacts with CEA and is unreactive with normalcross-reactive antigen (NCA) and meconium antigen (MA). However,antibodies against these cross-reactive determinants may be used incombination therapy with CEA-specific antibodies, such as combined withthe MN-14 monoclonal antibody.

In another embodiment of the present invention, the MN-14 monoclonalantibody or fragment thereof is a humanized or fully human MN-14antibody or fragment thereof. The framework regions (FRs) of the lightand heavy chain variable regions of the humanized MN-14 antibody orfragment thereof preferably comprise at least one amino acid substitutedfrom the corresponding FRs of a murine MN-14 monoclonal antibody. Stillpreferred, the humanized MN-14 antibody or fragment thereof comprises atleast one amino acid from the corresponding FR of the murine MN-14antibody selected from the group consisting of amino acid residue 24,28, 30, 48, 49, 74 and 94 of the murine heavy chain variable region(KLHuVhAIGA) of FIG. 14A-C as noted above. The amino acid sequence of apreferred humanized heavy chain variable region is also set forth inHansen et al., U.S. Pat. No. 5,874,540, which is incorporated byreference in its entirety. Also preferred, the humanized heavy chainvariable region comprises the amino acid sequence set forth in FIGS.14A-C, designated as KLHuVhAIG and KLHuVhAIGAY. In another embodiment,the humanized MN-14 antibody or fragment thereof comprises at least oneamino acid from the corresponding FR of the murine MN-14 light chainvariable region. Most preferably, the humanized MN-14 antibody orfragment thereof comprises the light chain variable region of FIG. 13Aor FIG. 22A or FIG. 23A. Another embodiment of the present invention isa composition comprising a chimeric MN-14 monoclonal antibody orfragment thereof and at least one therapeutic agent, which are notconjugated to each other, and thus are present in the composition asunconjugated forms of each of the components. Preferably, the chimericMN-14 antibody or fragment thereof comprises the CDRs of the murine MN14light chain variable region set forth in FIG. 13A or FIG. 22A or FIG.23A and the CDRs of the murine MN14 heavy chain variable region as setforth in FIGS. 14A-C or FIG. 22B or FIG. 23B.

Also described herein is a composition comprising a naked murine,humanized, chimeric or human Class III anti-CEA antibody or fragmentthereof and a therapeutic agent, and a second naked or conjugated ClassIII anti-CEA antibody or antibody fragment thereof, that isnon-blocking, i.e., does not block binding of the first Class IIIanti-CEA antibody or fragment thereof, and formulated in apharmaceutically acceptable vehicle. In other words, both Class IIIanti-CEA antibodies or fragments thereof are non-blocking to each other,thus, allowing both antibodies or fragments thereof to bind to CEA(CD66e). Additionally, the Class III CEA antibody or antibody fragmentof the present invention, as well as those for use in combinationtherapy, do not bind granulocytes or CD66a-d. Other Class III antibodiessuitable for combination therapy as a naked antibody or as a componentof an immunoconjugate, with the naked Class III anti-CEA antibody ofantibody fragment of the present invention include the non-blockingantibodies or fragments thereof described in Kuroki et al., JP J. CancerRes., 78(4):386 (1987) and Hammarstrom (Cancer Res. 52(8):2329 (1992)),that also do not bind granulocytes or CD66a-d.

Additionally, other anti-CEA antibodies, such as Class II or Class Ianti-CEA antibodies, can be used in combination with the Class IIIanti-CEA antibody of the present invention, in either a naked orconjugated form. Such Class II antibodies or antibody fragments that canbe used for combination therapy are non-blocking and do not bindgranulocytes or CD66a-d but are reactive with meconium antigen (MA) andCEA. For example, one or more chimeric or humanized Class II anti-CEAantibody or fragment thereof, such as MN-6 or NP-3, may be combined witha Class III anti-CEA antibody or fragment thereof of the presentinvention. These two antibodies do not react with CD66a-d or withgranulocytes (Hansen et al., Cancer Jun. 1, 1993; 71(11):3478-85). Anumber of publications disclose MAbs that recognize CEA and differentmembers of the CEA gene family, such as Thompson et al., J. Clin. Lab.Anal. 5:344 (1991); Kuroki et al., J. Biol. Chem. 266:11810 (1991);Nagel et al., Eur. J. Biochem. 214:27 (1993); Skubitz et al., J.Immunol. 155:5382 (1995); Skubitz et al., J. Leukoc. Biol. 60:106(1996); and Chen et al., Proc. Natl. Acad. Sci. USA 93:14851 (1996).

Moreover, the second antibody or antibody fragment is eitherunconjugated (naked) or conjugated to at least one therapeutic agent(immunoconjugate). Immunoconjugates can be prepared by indirectlyconjugating a therapeutic agent to an antibody component. Generaltechniques are described in Shih et al., Int. J. Cancer, 41:832 (1988);Shih et al., Int. J. Cancer, 46:1101 (1990); and Shih et al., U.S. Pat.No. 5,057,313. The general method involves reacting an antibodycomponent having an oxidized carbohydrate portion with a carrier polymerthat has at least one free amine function and that is loaded with aplurality of drug, toxin, chelator, boron addends, or other therapeuticagent. This reaction results in an initial Schiff base (imine) linkage,which can be stabilized by reduction to a secondary amine to form thefinal conjugate. Preferably, the anti-CEA antibody or fragment thereofin the composition for treatment is a MN-14 antibody or fragmentthereof. More preferred, the MN-14 antibody or fragment thereof ishumanized.

Also contemplated in the present invention is a composition comprising anaked humanized, chimeric, murine or human Class III anti-CEA antibodyor fragment thereof and a therapeutic agent, and a conjugated orunconjugated second antibody or antibody fragment thereof. In oneembodiment, the second antibody or fragment thereof is unconjugated(naked) or conjugated to at least one therapeutic agent. Non Class I,Class II or Class III anti-CEA antibodies and fragments thereof that aresuitable for combination therapy include, but are not limited to,carcinoma-associated antibodies and fragments thereof. Examples ofcarcinoma associated antibodies and antibody fragments bind EGP-1, EGP-2(e.g., 17-1A), MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR,HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn, Thomson-Friedenreichantigens, tumor necrosis antigens, VEGF, P1GF or other tumorangiogenesis antigens, Ga 733, IL-6, insulin-like growth factor-1,tenascin, fibronectin or a combination thereof. As discussed supra,non-blocking Class II and Class III anti-CEA MAbs that do not bindCD66a-d or granulocytes or alternatively, Class II anti-CEA MAbs that dobind CD66a, b and d or Class I anti-CEA MAbs that bind CD66a, b and d,as well as CD66c, may also be used in combination with Class III CEAantibodies. Other antibodies and antibody fragments suitable forcombination therapy also include those targeted against oncogene markersor products, or antibodies against tumor-vasculature markers, such asthe angiogenesis factor, Placental Growth Factor (P1GF), and antibodiesagainst certain immune response modulators, such as antibodies to CD40.

Methods

Also described in the present invention are methods for treatingmedullary thyroid carcinoma and non-medullary thyroid carcinomas.Non-medullary thyroid carcinomas include colorectal cancer and any otherCEA expressing tumor, such as pancreatic cancer, breast cancer,hepatocellular carcinoma, ovarian cancer, certain kinds of lung,head-and-neck, endometrial, bladder, and liver cancers that expressvariable quantities of CEA. The CEA levels in these types of cancers aremuch lower than present in medullary thyroid carcinomas but all that isnecessary is that the CEA levels be sufficiently high so that the ClassIII anti-CEA therapy provides an effective treatment. Normal colonmucosa has about 100-500 ng/gram but carcinomas expressing CEA at levelsof about 5 mcg/gram of tissue are suitable for treatment with themethods described in the instant invention.

For example, contemplated herein is a method for treating medullarythyroid carcinoma or non-medullary thyroid carcinoma comprisingadministering to a subject, either concurrently or sequentially, atherapeutically effective amount of a Class III anti-CEA monoclonalantibody or fragment thereof and at least one therapeutic agent, andoptionally formulated in a pharmaceutically acceptable vehicle.Preferably, the Class III anti-CEA monoclonal antibody or fragmentthereof is chimeric, murine, humanized or human, wherein the chimeric,humanized, murine, or human Class III anti-CEA MAb retains substantiallythe Class III anti-CEA binding specificity of the murine MAb. Morepreferably, the Class III anti-CEA antibody is humanized, and mostpreferably, the humanized MN-14 monoclonal antibody, as described hereinand in U.S. Pat. No. 5,874,540. Preferably the therapeutic agent is acytotoxic agent, more preferably an alkylating agent, and mostpreferably, dacarbazine (DTIC). But in another embodiment, thetherapeutic agent may also not be DTIC. Other classes of anti-cancercytostatic and cytotoxic agents, such as 5-fluorouracil, CPT-11 (whichis also known as irinotecan and camptosar) and oxaliplatin can also beused in combinations with these antibodies, especially in the therapy ofcolorectal cancers. In other cancer types, cancer drugs that are knownto be effective are also good candidates for combining with the antibodytherapies proposed herein.

Also contemplated herein is a method for treating medullary thyroidcarcinoma and non-medullary thyroid carcinoma comprising administeringto a subject, either concurrently or sequentially, a therapeuticallyeffective amount of a first Class III anti-CEA monoclonal antibody orfragment thereof and at least one therapeutic agent, and a naked orconjugated second humanized, chimeric, human or murine monoclonalantibody or fragment thereof, and optionally formulated in apharmaceutically acceptable vehicle. Preferably, the first Class IIIanti-CEA MAb is a humanized MN-14 antibody or fragment thereof. In oneembodiment, the second antibody or fragment thereof is acarcinoma-associated antibody or fragment thereof selected from thegroup consisting of a monoclonal antibody or fragment thereof reactivewith TAG-72, EGFR, HER2/neu, MUC1, MUC2, MUC3, MUC4, EGP-1, EGP-2, AFP,Tn, IL-6, insulin growth factor-1, or another such tumor-associatedantigen, as described above. In another embodiment, the second antibodyor fragment thereof can be a different Class III anti-CEA antibody orfragment thereof that is non-blocking and does not bind granulocytes orCD66a-d.

In another embodiment, the second anti-CEA antibody is a Class IIantibody or fragment thereof, such as those described in Hammarstrom andKuroki, provided that they do not bind granulocytes or CD66a-d. Inanother embodiment, this antibody includes Class I MAbs or fragmentsthereof, that react with CD66a, b, or d as well as CD66c. The antibodiesand fragments thereof may be administered either concurrently orsequentially with each other or the therapeutic agent. In oneembodiment, the second antibody or fragment thereof is either naked orconjugated to a therapeutic agent.

Accordingly, the present invention contemplates the administration ofnaked murine, humanized, chimeric and human anti-CEA antibodies andfragments thereof sequentially or concurrently with one or moretherapeutic agents, or administered as a multimodal therapy. A ClassIII, anti-CEA antibody is preferred but any anti-CEA antibody thattargets tumor cells are useful in the present invention. A naked ClassIII anti-CEA antibody as described herein can significantly increase thechemosensitivity of cancer cells to one or more therapeutic agents. Forexample, treatment of colon cancer cells with a naked Class III,anti-CEA antibody, MN-14 as described herein, either before orconcurrently with a therapeutic agent, such as CPT-11, 5′-fluorouracil(5-FU) or oxaliplatin, improves a cell's response to a therapeuticagent, such as a cytotoxic drug. Further, these therapeutic methods oftreatment with a naked Class III, anti-CEA antibody alone or incombination with a therapeutic agent can be further enhanced byadministering an immunomodulator as described herein, prior to theadministration of the naked antibody or the administration of the nakedantibody and at least one of the therapeutic agents.

Multimodal therapies of the present invention include immunotherapy witha Class III anti-CEA antibody or fragment thereof, and a therapeuticagent, supplemented with administration of an unconjugated or conjugatedantibody, unconjugated or conjugated fusion protein, or fragmentthereof. For example, an unconjugated humanized, chimeric, murine orhuman MN-14 MAb or fragment thereof may be combined with another nakedhumanized, murine, chimeric or human Class III anti-CEA antibody (suchas an antibody against a different epitope on CEA and also does not bindgranulocytes or CD66a-d), or a humanized, chimeric, murine or humanClass III anti-CEA antibody immunoconjugate conjugated to aradioisotope, chemotherapeutic agent, cytokine, enzyme,enzyme-inhibitor, hormone or hormone antagonist, metal, toxin, antisenseoligonucleotide (e.g., anti-bc1-2), or a combination thereof. A nakedClass III anti-CEA antibody or fragment thereof may also be combinedwith a conjugated or unconjugated fusion protein of a murine, humanized,chimeric or human Class III anti-CEA antibody. However, the Class IIIanti-CEA antibodies for combination therapy are non-blocking to eachother and unable to bind granulocytes or CD66a-d. Preferably, the nakedClass III anti-CEA antibody is administered sequentially or concurrentlywith the second naked or conjugated antibody, fusion protein, orfragment thereof. Also preferred, one of the antibodies or antibodyfragments for use in combination therapy is a naked humanized MN-14antibody or fragment thereof. Additionally, the second antibody used asa naked or conjugated antibody, fusion protein, or fragment thereof, maybe a human, humanized, chimeric or murine Class II CEA antibody orfragment thereof that is non-blocking and does not bind granulocytes orCD66a-d. A preferred combination of antibodies according to thisembodiment would include a naked cross reactive anti-CD66a-d antibodywhich lacks an effector function, which does not activate complement anddoes not induce cytokine release. In addition, a cross reactiveanti-CD66a-d Fab′ or even a F(ab′)₂ would likely not damage granulocytesand could be used with a Class III anti-CEA MAb or a Class II anti-CEAMAb of the NP-3 type.

In the methods described herein, subjects receive at least one nakedClass III anti-CEA antibody or fragment thereof, administered before,after or in conjunction with a therapeutic agent. In one embodiment, aClass III anti-CEA antibody is used for pretreating cells, i.e.,administered before a therapeutic agent. Preferably, the class IIIanti-CEA antibody is an MN-14 antibody, such as a humanized MN-14(hMN-14) that is administered at least one hour before a therapeuticagent, such as 5-FU or CPT-11.

Preferably, the therapeutic agent is a drug used in standard cancerchemotherapy, such as taxane or platinum drugs in ovarian cancer,fluorouracil, CPT-11, and oxaliplatin drugs in colorectal cancer,gemcitabine in pancreatic and other cancers, or taxane derivatives inbreast cancers. COX-2 inhibitors represent still another class of agentsthat show activity in combination with typical cytotoxic agents incancer chemotherapy, and can be used in this invention in the same way,but combined in addition with CEA antibodies alone and in combinationwith other cancer-associated antibodies. Optionally, these drugs can beused in combination with radiolabeled antibodies, either CEA antibodyconjugates or radioconjugates with other carcinoma-associatedantibodies, of the kinds described above. Also preferred, the Class IIIanti-CEA antibody or fragment thereof is a MN-14 antibody or fragmentthereof. Still preferred, the MN-14 antibody or fragment thereof ishumanized.

In a preferred embodiment, a naked Class III anti-CEA antibody orfragment thereof is administered sequentially (either prior to or after)or concurrently with dacarbazine (DTIC), doxorubin, cyclophosphamide orvincristine, or any combination of these. For example, DTIC andcyclophosphamide may be administered sequentially or concurrently with anaked Class III anti-CEA antibody or fragment thereof. Preferably, theanti-CEA antibody or fragment thereof is a humanized MN-14 antibody orfragment thereof. Similarly, 5-fluorouracil in combination with folinicacid, alone or in combination irinotecan (CPT-11) or in combination withoxaliplatin, is a regimen used to treat colorectal cancer. Othersuitable combination chemotherapeutic regimens are well known, such aswith oxaliplatin alone, or in combination with these other drugs, tothose of skill in the art. Accordingly, combination therapy with any ofthese chemotherapeutic agents and a naked Class III anti-CEA antibody orfragment thereof can be used to treat MTC or non-MTC, depending on theregimen used. In medullary thyroid carcinoma, still otherchemotherapeutic agents may be preferred, such as one of the alkylatingagents (e.g., DTIC), as well as gemcitabine and other more recentclasses of cytotoxic drugs. The chemotherapeutic drugs and a naked ClassIII anti-CEA antibody or fragment thereof, can be administered in anyorder, or together. In other words, the antibody and therapeutic agentmay be administered concurrently or sequentially. In a preferredmultimodal therapy, both chemotherapeutic drugs and naked Class IIIanti-CEA antibodies or fragments thereof are administered before, after,or co-administered with a conjugated or unconjugated anti-CEA antibody,fusion protein, or fragment thereof, according to the present invention.Preferably, the Class III anti-CEA antibody or fragment thereof is ahumanized MN-14 antibody or fragment thereof.

A preferred treatment schedule of multimodal treatment is administeringboth hMN-14 and DTIC for 3 days, and administering only hMN-14 on days7, 14, 21 and then every 21 days for a treatment duration of 12 months.The doses of hMN-14 are 0.5-15 mg/kg body weight per infusion, morepreferably 2-8, and still more preferably 3-5 mg/kg per infusion, andthe doses of DTIC are as currently applied at the preferred doseclinically, but could also be given at two-thirds or less of the maximumpreferred dose in use, thereby decreasing drug-related adverse events.Repeated drug cycles can be given, such as every 1-6 months, withcontinuation of the naked antibody therapy, or with different schedulesof radiolabeled antibody, drug-conjugated antibody, and inclusion ofcertain cytokines, such as G-CSF and/or GM-CSF, each dose adjusted sothat toxicity to the patient is not enhanced by the therapeuticcombination. The application of a cytokine growth factor, such as G-CSF,may enable even higher doses of myelosuppressive agents, such asradiolabeled antibody or cytotoxic drugs, to be administered, and theseschedules and doses will be adjusted for the patients individually,depending on their disease status and prior therapy, all influence bonemarrow status and tolerability to additional cytotoxic therapies. In apreferred embodiment, the MN-14 antibody or fragment thereof isadministered in a dosage of 100-600 milligrams protein per dose perinjection. Still preferred, the MN-14 antibody or fragment thereof isadministered in a dosage of 300-400 milligrams of protein per dose perinjection, with repeated doses preferred. The preferred antibodyschedule is infusing once weekly or even less frequently, such as onceevery other week or even every third week, depending on a number offactors, including the extent of the disease and the amount of CEAcirculating in the patient's blood.

Therapeutic Aunts

The therapeutic agents recited here are those agents that also areuseful for administration separately with a naked antibody, as describedherein. Suitable therapeutic agents can be selected from the groupconsisting of a cytotoxic agent, a toxin, a hormone, a radionuclide, animmunomodulator, a photoactive therapeutic agent (such as a chromagen ordye), an antisense oligonucleotide, an immunoconjugate, another nakedantibody, a hormone, or a combination thereof. Therapeutic agentsinclude, for example, chemotherapeutic drugs such as vinca alkaloids andother alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, COX-2 inhibitors,antimitotics, antiangiogenic and apoptotoic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others fromthese and other classes of anticancer agents, and the like. Other usefulcancer chemotherapeutic drugs for the preparation of immunoconjugatesand antibody fusion proteins include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, oxaliplatin, folic acid analogs,COX-2 inhibitors, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, toxins (e.g., RNAse, Pseudomonasexotoxin), and the like. Preferred therapeutic agents include DTIC,CPT-11, 5-fluorouracil, taxol, oxaliplatin, doxorubicin,cyclophosphamide and vincristine, or a combination thereof, depending onthe malignancy to be treated. Suitable chemotherapeutic agents aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

A toxin, such as Pseudomonas exotoxin, may also be administered with anaked Class III anti-CEA antibody or fragment thereof. Preferably, theClass III anti-CEA antibody or fragment thereof is a humanized MN-14antibody or fragment thereof. Other suitable microbial, plant or animaltoxins to be administered unconjugated to, but before, after, orsimultaneously with the naked Class III anti-CEA antibody or fragmentthereof include ricin, abrin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See,for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, CA-ACancer Journal for Clinicians 44:43 (1994). Additional toxins suitablefor use in the present invention are known to those of skill in the artand are disclosed in U.S. Pat. No. 6,077,499, which is incorporated inits entirety by reference. These can be derived, for example, fromanimal, plant and microbial sources, or chemically or recombinantlyengineered. The toxin can be a plant, microbial, or animal toxin, or asynthetic variation thereof.

An immunomodulator, such as a cytokine may also be administeredunconjugated to the chimeric, murine, humanized or human Class IIIanti-CEA antibody or fragment thereof of the present invention. As usedherein, the term “immunomodulator” includes cytokines, stem cell growthfactors, lymphotoxins, such as tumor necrosis factor (TNF), andhematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1),IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulatingfactors (e.g., granulocyte-colony stimulating factor (G-CSF) andgranulocyte macrophage-colony stimulating factor (GM-CSF)), interferons(e.g., interferons-α, -β and -γ), the stem cell growth factor designated“S1 factor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21,interferon-γ, TNF-α, and the like. Therefore, subjects can receive anaked Class III anti-CEA antibody or fragment thereof and a separatelyadministered cytokine, which can be administered before, concurrently orafter administration of the naked Class III anti-CEA antibody orfragment thereof. Since some antigens may also be immunomodulators, CD40antigen, for example, may also be administered in combination with anaked Class III anti-CEA antibody or fragment thereof either together,before or after the naked antibody or antibody combinations areadministered. Additionally, radionuclides suitable for treating adiseased tissue include, but are not limited to, ³²P, ³³P, ⁴⁷Sc, ⁵⁹Fe,⁶⁴Cu, ⁶⁷Cu, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁰Y, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ¹²⁵I,¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb, ²¹²Pb, and ²¹³Bi, ⁵⁸Co, ⁶⁷Ga,^(80m)Br, ^(99m)Tc, ^(103m)Rh, ¹⁰⁹Pt, ¹¹¹In, ¹¹⁹Sb, ¹⁶¹Ho, ^(189m)Os,¹⁹²Ir, ¹⁵²Dy, ²¹¹At, ²¹²Bi, ²²³Ra, ²¹⁹Rn, ²¹⁵Po, ²¹¹Bi, ²²⁵Ac, ²²¹Fr,²¹⁷At, ²¹³Bi, ⁸⁸Y and ²⁵⁵Fm. Preferred radionuclides are ¹²⁵I, ¹³¹I,⁹⁰Y, ¹⁷⁷Lu, and ²²⁵Ac. Also preferred, the radionuclide has an energybetween 20 and 10,000 keV.

Pharmaceutically Acceptable Vehicles

The naked murine, humanized, chimeric and human Class III anti-CEA MAbsto be delivered to a subject can comprise one or more pharmaceuticallyacceptable vehicles, one or more additional ingredients, or somecombination of these.

The unconjugated Class III anti-CEA antibodies and fragments thereof ofthe present invention can be formulated according to known methods toprepare pharmaceutically useful compositions. Preferably, the Class IIIanti-CEA antibody or fragment thereof is a MN-14 antibody or fragmentthereof. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable vehicle. Other acceptable vehicles arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The unconjugated Class III anti-CEA antibody or fragment thereof of thepresent invention can be formulated for intravenous administration via,for example, bolus injection or continuous infusion. Preferably, theClass III anti-CEA antibody or fragments is a MN-14 antibody or fragmentthereof. Formulations for injection can be presented in unit dosageform, e.g., in ampules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Additional pharmaceutical methods may be employed to control theduration of action of the agent and naked antibody or fragment thereof.Control release preparations can be prepared through the use of polymersto complex or adsorb the naked antibody. For example, biocompatiblepolymers include matrices of poly(ethylene-co-vinyl acetate) andmatrices of a polyanhydride copolymer of a stearic acid dimer andsebacic acid. Sherwood et al., Bio/Technology 10:1446 (1992). The rateof release of an antibody or fragment thereof from such a matrix dependsupon the molecular weight of the immunoconjugate or antibody, the amountof antibody within the matrix, and the size of dispersed particles.Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al., supra.Other solid dosage forms are described in Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

The unconjugated Class III anti-CEA antibody or fragment thereof mayalso be administered to a mammal subcutaneously or even by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. In general, the dosage of anadministered naked antibody or fragment thereof for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of naked antibody orfragment thereof that is in the range of from about 0.5 mg/kg to 20mg/kg as a single intravenous infusion, although a lower or higherdosage also may be administered as circumstances dictate. This dosagemay be repeated as needed, for example, once per month for 4-10 months,preferably once per every other week for 16 weeks, and more preferably,once per week for 8 weeks. It may also be given less frequently, such asevery other week for several months or given more frequently and/or overa longer duration. The dosage may be given through various parenteralroutes, with appropriate adjustment of the dose and schedule.

For purposes of therapy, the Class III anti-CEA antibody or fragmentthereof is administered to a mammal in a therapeutically effectiveamount to reduce the size of the tumor as compared to untreatedcontrols. Preferably, the Class III anti-CEA antibody or fragmentthereof is a humanized MN-14 antibody or fragment thereof. A suitablesubject for the present invention is usually a human, although anon-human mammal or animal subject is also contemplated. An antibodypreparation is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient mammal. Inparticular, an antibody preparation of the present invention isphysiologically significant if its presence invokes an antitumorresponse. A physiologically significant effect could also be theevocation of a humoral and/or cellular immune response in the recipientmammal.

The present invention further includes the following numberedembodiments:

A composition comprising at least one anti-CEA monoclonal antibody (MAb)or fragment thereof and at least one therapeutic agent. The compositionof embodiment 1, wherein said anti-CEA MAb is a Class I, Class II orClass III anti-CEA MAb, and when said MAb is a Class I or Class II MAband is reactive with granulocytes, said MAb is a monovalent form of theMAb.

2. The composition of embodiment 1, wherein said anti-CEA MAb orfragment thereof is humanized, wherein said humanized MAb retainssubstantially the anti-CEA binding specificity of a murine anti-CEA MAb.

3. The composition of embodiment 1, wherein said anti-CEA MAb orfragment thereof is a chimeric MAb, and wherein said chimeric MAbretains substantially the anti-CEA binding specificity of murineanti-CEA MAb.

4. The composition of embodiment 1, wherein said anti-CEA MAb orfragment thereof is a fully human MAb, and wherein said fully human MAbretains substantially the anti-CEA binding specificity of murineanti-CEA MAb.

5. The composition of embodiment 1, wherein said anti-CEA monoclonalantibody or fragment thereof is a MN-14 antibody or fragment thereof.

6. The composition of embodiment 5, wherein said MN-14 monoclonalantibody or fragment thereof comprises the complementarity-determiningregions (CDRs) of a murine MN-14 monoclonal antibody, wherein the CDRsof the light chain variable region of said MN-14 antibody comprises CDR1comprising the amino acid sequence KASQDVGTSVA (SEQ ID NO: 20); CDR2comprising the amino acid sequence WTSTRHT (SEQ ID NO: 21); and CDR3comprising the amino acid sequence QQYSLYRS (SEQ ID NO: 22); and theCDRs of the heavy chain variable region of said anti-CEA antibodycomprises CDR1 comprising TYWMS (SEQ ID NO: 23); CDR2 comprisingEIHPDSSTINYAPSLKD (SEQ ID NO: 24); and CDR3 comprising LYFGFPWFAY (SEQID NO: 25).

7. The composition of embodiment 1, wherein said anti-CEA monoclonalantibody reacts with CEA and is unreactive with normal cross-reactiveantigen (NCA) and meconium antigen (MA).

8. The composition of embodiment 7, wherein said MN-14 monoclonalantibody or fragment thereof is a humanized MN-14 antibody or fragmentthereof.

9. The composition of embodiment 7, wherein said MN-14 monoclonalantibody or fragment thereof is a chimeric MN-14 antibody or fragmentthereof.

10. The composition of embodiment 7, wherein said MN-14 monoclonalantibody or fragment thereof is a fully human MN-14 antibody or fragmentthereof.

11. The composition of embodiment 8, wherein the framework regions (FRs)of the light and heavy chain variable regions of said humanized MN-14antibody or fragment thereof comprise at least one amino acidsubstituted from the corresponding FRs of a murine MN-14 monoclonalantibody.

12. The composition of embodiment 11, wherein said humanized MN-14antibody or fragment thereof comprises at least one amino acid from saidcorresponding FR of said murine MN-14 antibody is selected from thegroup consisting of amino acid residue 24, 28, 30, 48, 49, 74 and 94 ofthe murine heavy chain variable region (KLHuVhAIGA) of FIG. 14A-C orFIG. 22B (hMn-14) or FIG. 23B.

13. The composition of embodiment 11, wherein said humanized MN-14antibody or fragment thereof comprises at least one amino acid from saidcorresponding FR of said murine MN-14 light chain variable region.

14. The composition of embodiment 8, wherein said humanized MN-14antibody or fragment thereof comprises the light chain variable regionas set forth in FIG. 13A or FIG. 22A or FIG. 23A, and the heavy chainvariable region set forth in FIG. 14A-C designated as KLHuVhAIGA or FIG.22B (hMN-14) or FIG. 23B.

15. The composition of embodiment 9, wherein said chimeric MN-14antibody or fragment thereof comprises the light chain variable regionas set forth in FIG. 13A designated as murine MN-14 VK and the heavychain variable region set forth in FIG. 14A-C designated as murine MN-14VH.

16. The composition of any of embodiments 1-15, wherein said fragment isselected from the group consisting of F(ab′)₂, Fab′, Fab, Fv and scFv.

17. The composition of any of embodiments 1-15, wherein said therapeuticagent is selected from the group consisting of a naked antibody, acytotoxic agent, a drug, a radionuclide, an immunomodulator, aphotoactive therapeutic agent, an immunoconjugate, a hormone, a toxin,an antisense oligonucleotide, or a combination thereof, optionallyformulated in a pharmaceutically acceptable vehicle.

18. The composition of embodiment 17, wherein said combination thereofcomprises vincristine, doxorubicin, oxaliplatin, CPT-11, fluorouracil,DTIC and cyclophosphamide.

19. The composition of embodiment 17, wherein said therapeutic agent isa naked antibody or an immunoconjugate.

20. The composition of embodiment 19, wherein said naked antibody or anantibody portion of said immunoconjugate comprises a humanized,chimeric, human or murine monoclonal antibody or fragment thereofselected from the group consisting of a monoclonal antibody or fragmentthereof reactive with EGP-1, EGP-2 (e.g., 17-1A), MUC-1, MUC-2, MUC-3,MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM,AFP, Tn, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF,P1GF, or other tumor angiogenesis antigens, Ga 733, IL-6, insulin-likegrowth factor-1, tenascin, fibronectin or a combination thereof.

21. The composition of embodiment 20, wherein said fragment is selectedfrom the group consisting of F(ab)₂, F(ab′)₂, Fab′, Fab, Fv and scFv.

22. The composition of any of embodiments 1-15, wherein said therapeuticagent is not DTIC.

23. A method for treating non-medullary thyroid carcinoma comprisingadministering to a subject, either concurrently or sequentially, atherapeutically effective amount of an anti-CEA antibody or fragmentthereof and at least one therapeutic agent, and optionally formulated ina pharmaceutically acceptable vehicle.

24. The method of embodiment 23, wherein said anti-CEA MAb or fragmentthereof is humanized, wherein said humanized MAb retains substantiallythe anti-CEA binding specificity of a murine anti-CEA MAb.

25. The method of embodiment 23, wherein said anti-CEA MAb or fragmentthereof is a chimeric MAb, and wherein said chimeric MAb retainssubstantially the anti-CEA binding specificity of murine anti-CEA MAb.

26. The method of embodiment 23, wherein said anti-CEA monoclonalantibody or fragment thereof is a MN-14 antibody or fragment thereof.

27. The method of embodiment 23, wherein said MN-14 monoclonal antibodyor fragment thereof comprises the complementarity-determining regions(CDRs) of a murine MN-14 monoclonal antibody, wherein the CDRs of thelight chain variable region of said MN-14 antibody comprises CDR1comprising the amino acid sequence KASQDVGTSVA (SEQ ID NO: 20); CDR2comprising the amino acid sequence WTSTRHT (SEQ ID NO: 21); and CDR3comprising the amino acid sequence QQYSLYRS (SEQ ID NO: 22); and theCDRs of the heavy chain variable region of said anti-CEA antibodycomprises CDR1 comprising TYWMS (SEQ ID NO: 23); CDR2 comprisingEIHPDSSTINYAPSLKD (SEQ ID NO: 24); and CDR3 comprising LYFGFPWFAY (SEQID NO: 25).

28. The method of embodiment 27, wherein said MN-14 monoclonal antibodyreacts with CEA and is unreactive with normal cross-reactive antigen(NCA) and meconium antigen (MA).

29. The method of embodiments 28, wherein said MN-14 monoclonal antibodyor fragment thereof is a humanized MN-14 antibody or fragment thereof.

30. The method of embodiments 28, wherein said MN-14 monoclonal antibodyor fragment thereof is a chimeric MN-14 antibody or fragment thereof.

31. The method of embodiments 28, wherein said MN-14 monoclonal antibodyor fragment thereof is a fully human MN-14 antibody or fragment thereof.

32. The method of embodiment 29, wherein the framework regions (FRs) ofthe light and heavy chain variable regions of said humanized MN-14antibody or fragment thereof comprise at least one amino acidsubstituted from the corresponding FRs of a murine MN-14 monoclonalantibody.

33. The method of embodiment 32, wherein said humanized MN-14 antibodyor fragment thereof comprising at least one amino acid from saidcorresponding FR of said murine MN-14 antibody is selected from thegroup consisting of amino acid residue 24, 28, 30, 48, 49, 74 and 94 ofthe murine heavy chain variable region of FIG. 14A-C designated asKLHuVhAIGA or FIG. 22B (hMN-14) or FIG. 23B.

34. The method of embodiment 32, wherein said humanized MN-14 antibodyor fragment thereof comprising at least one amino acid from saidcorresponding FR of said murine MN-14 light chain variable region.

35. The method of embodiment 32, wherein said humanized MN-14 antibodyor fragment thereof comprises the light chain variable region as setforth in FIG. 13A or FIG. 22A (hMN-14) or FIG. 23A and the heavy chainvariable region set forth in FIG. 14A-C designated as KLHuVhAIGA or FIG.22B (hMN-14) or FIG. 23B.

36. The method of any of embodiments 23-35, wherein said fragment isselected from the group consisting of F(ab)₂, F(ab′)₂, Fab′, Fab, Fv andsFv.

37. The method of any of embodiments 23-35, wherein said therapeuticagent is selected from the group consisting of humanized, chimeric,human or murine monoclonal antibody or fragment thereof selected fromthe group consisting of a Class I anti-CEA monoclonal antibody, Class IIanti-CEA monoclonal antibody, Class III anti-CEA monoclonal antibody,and a fragment thereof, and is administered either concurrently orsequentially in a therapeutically effective amount.

38. The method of embodiment 37, wherein said antibody or fragmentthereof is either naked or conjugated to another therapeutic agent.

39. The method of any of embodiments 23-35, wherein said therapeuticagent is selected from the group consisting of a naked antibody,cytotoxic agent, a drug, a radionuclide, an immunomodulator, aphotoactive therapeutic agent, an antisense oligonucleotide, animmunoconjugate of a CEA or non-CEA antibody, a hormone, or acombination thereof, optionally formulated in a pharmaceuticallyacceptable vehicle.

40. The method of embodiment 39, wherein said therapeutic agent isselected from the group consisting of a humanized, chimeric, human ormurine monoclonal antibody or fragment thereof reactive with EGP-1,EGP-2 (e.g., 17-1A), IL-6, MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4,TAG-72, EGFR, EGP-2, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, P1GF orother tumor angiogenesis antigens, Ga 733, IL-6, insulin-like growthfactor-1, and a combination thereof, and is administered to said subjecteither concurrently or sequentially in a therapeutically effectiveamount.

41. The method of embodiment 40, wherein said antibody or fragmentthereof is either naked or conjugated to another therapeutic agent.

42. The method of any of embodiments 23-35, wherein said therapeuticagent is not DTIC.

43. The method of embodiment 39, wherein said cytotoxic agent is a drugor a toxin.

44. The method of embodiment 43, wherein said drug possesses thepharmaceutical property selected from the group consisting ofantimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,alkaloid, COX-2, and antibiotic agents and combinations thereof.

45. The method of embodiment 43, wherein said drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,antimetabolites, antibiotics, enzymes, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, antagonists,endostatin, taxols, camptothecins, doxorubicins and their analogs, and acombination thereof.

46. The method of embodiment 43, wherein said toxin is a microbial,plant or animal toxin selected from the group consisting of ricin,abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

47. The method of embodiment 39, wherein said immunomodulator isselected from the group consisting of a cytokine, a stem cell growthfactor, a lymphotoxin, a hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), a stem cell growth factor,erythropoietin, thrombopoietin and a combination thereof.

48. The method of embodiment 47, wherein said lymphotoxin is tumornecrosis factor (TNF), said hematopoietic factor is an interleukin (IL),said colony stimulating factor is granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF)),said interferon is interferons-α, -β or -γ, and said stem cell growthfactor is designated “S1 factor”.

49. The method of embodiment 47, wherein said immunomodulator comprisesIL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-γ, TNF-αor a combination thereof.

50. The method of embodiment 39, wherein said radionuclide has an energybetween 20 and 10,000 keV.

51. The method of embodiment 50, wherein said radionuclide is selectedfrom the group consisting of ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁸Y, ²²⁵Ac, ¹⁷⁷Lu, ¹⁸⁸Re,¹⁸⁶R, and combinations thereof.

52. The method of embodiment 39, wherein said photoactive therapeuticagent is a chromogen or dye.

53. The method of embodiment 44, wherein said alkylating agent isdacarbazine.

54. The method of embodiment 53, wherein said MN-14 antibody or fragmentthereof is administered in a dosage of 100 to 600 milligrams protein perdose per injection.

55. The method of embodiment 54, wherein said MN-14 antibody or fragmentthereof is administered in a dosage of 300-400 milligrams protein perdose per injection.

56. A method for treating medullary thyroid carcinoma comprisingadministering to a subject, either concurrently or sequentially, atherapeutically effective amount of an anti-CEA monoclonal antibody orfragment thereof and at least one therapeutic agent, and optionallyformulated in a pharmaceutically acceptable vehicle.

57. The method of embodiment 56, wherein said anti-CEA MAb or fragmentthereof is humanized, wherein said humanized MAb retains substantiallythe anti-CEA binding specificity of a murine anti-CEA MAb.

58. The method of embodiment 56, wherein said anti-CEA MAb or fragmentthereof is a chimeric MAb, and wherein said chimeric MAb retainssubstantially the anti-CEA binding specificity of murine anti-CEA MAb.

59. The method of embodiment 56, wherein said anti-CEA monoclonalantibody or fragment thereof is a MN-14 antibody or fragment thereof.

60. The method of embodiment 59, wherein said MN-14 monoclonal antibodyor fragment thereof comprises the complementarity-determining regions(CDRs) of a murine MN-14 monoclonal antibody, wherein the CDRs of thelight chain variable region of said MN-14 antibody comprises CDR1comprising the amino acid sequence KASQDVGTSVA (SEQ ID NO: 20); CDR2comprising the amino acid sequence WTSTRHT (SEQ ID NO: 21); and CDR3comprising the amino acid sequence QQYSLYRS (SEQ ID NO: 22); and theCDRs of the heavy chain variable region of said Class III anti-CEAantibody comprises CDR1 comprising TYWMS (SEQ ID NO: 23); CDR2comprising EIHPDSSTINYAPSLKD (SEQ ID NO: 24); and CDR3 comprisingLYFGFPWFAY (SEQ ID NO: 25).

61. The method of embodiment 60, wherein said MN-14 monoclonal antibodyreacts with CEA and is unreactive with normal cross-reactive antigen(NCA) and meconium antigen (MA).

62. The method of embodiments 61, wherein said MN-14 monoclonal antibodyor fragment thereof is a humanized MN-14 antibody or fragment thereof.

63. The method of embodiments 61, wherein said MN-14 monoclonal antibodyor fragment thereof is a chimeric MN-14 antibody or fragment thereof.

64. The method of embodiments 61, wherein said MN-14 monoclonal antibodyor fragment thereof is a fully human MN-14 antibody or fragment thereof.

65. The method of embodiment 62, wherein the framework regions (FRs) ofthe light and heavy chain variable regions of said humanized MN-14antibody or fragment thereof comprise at least one amino acidsubstituted from the corresponding FRs of a murine MN-14 monoclonalantibody.

66. The method of embodiment 65, wherein said humanized MN-14 antibodyor fragment thereof comprising at least one amino acid from saidcorresponding FR of said murine MN-14 antibody is selected from thegroup consisting of amino acid residue 24, 28, 30, 48, 49, 74 and 94 ofthe murine heavy chain variable region of FIG. 14A-C or 22B.

67. The method of embodiment 65, wherein said humanized MN-14 antibodyor fragment thereof comprising at least one amino acid from saidcorresponding FR of said murine MN-14 light chain or heavy chainvariable region.

68. The method of embodiment 65, wherein said humanized MN-14 antibodyor fragment thereof comprises the light chain variable region as setforth in FIG. 13A or 22A (hMN-14) or 23A and the heavy chain variableregion set forth in FIG. 14A-C or 22B (hMN-14) or 23B.

69. The method of any of embodiments 56-68, wherein said fragment isselected from the group consisting of F(ab)₂, F(ab′)₂, Fab′, Fab, Fv andsFv.

70. The method of any of embodiments 56-68, wherein said therapeuticagent is selected from the group consisting of humanized, chimeric,human or murine monoclonal antibody or fragment thereof selected fromthe group consisting of a Class I anti-CEA monoclonal antibody, Class IIanti-CEA monoclonal antibody, Class III anti-CEA monoclonal antibody,and a fragment thereof, and is administered either concurrently orsequentially in a therapeutically effective amount.

71. The method of embodiment 70, wherein said antibody or fragmentthereof is either naked or conjugated to another therapeutic agent.

72. The method of any of embodiments 56-68, wherein said therapeuticagent is selected from the group consisting of a naked antibody,cytotoxic agent, a drug, a toxin, a radionuclide, an immunomodulator, anantisense oligonucleotide, a photoactive therapeutic agent, animmunoconjugate of a CEA or non-CEA antibody, a hormone, or acombination thereof, optionally formulated in a pharmaceuticallyacceptable vehicle.

73. The method of embodiment 72, wherein said therapeutic agent isselected from the group consisting of a humanized, chimeric, human ormurine monoclonal antibody or fragment thereof reactive with EGP-1,EGP-2 (e.g., 17-1A), MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72,EGFR, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, P1GF orother tumor angiogenesis antigens, Ga 733, IL-6, insulin-like growthfactor-1, and a combination thereof, and is administered to said subjecteither concurrently or sequentially in a therapeutically effectiveamount.

74. The method of embodiment 73, wherein said antibody or fragmentthereof is either naked or conjugated to another therapeutic agent.

75. The method of any of embodiments 56-68, wherein said therapeuticagent is not DTIC.

76. The method of embodiment 72, wherein said cytotoxic agent is a drugor a toxin.

77. The method of embodiment 72, wherein said drug possesses thepharmaceutical property selected from the group consisting ofantimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,alkaloid, COX-2, and antibiotic agents and combinations thereof.

78. The method of embodiment 76, wherein said drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,antimetabolites, antibiotics, enzymes, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, antagonists,endostatin, taxols, camptothecins, doxorubicins and their analogs, and acombination thereof.

79. The method of embodiment 76, wherein said microbial, plant or animaltoxin is selected from the group consisting of ricin, abrin, alphatoxin, saporin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

80. The method of embodiment 72, wherein said immunomodulator isselected from the group consisting of a cytokine, a stem cell growthfactor, a lymphotoxin, a hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), a stem cell growth factor,erythropoietin, thrombopoietin and a combination thereof.

81. The method of embodiment 80, wherein said lymphotoxin is tumornecrosis factor (TNF), said hematopoietic factor is an interleukin (IL),said colony stimulating factor is granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF)),said interferon is interferons-α, -β or -γ, and said stem cell growthfactor is designated “S1 factor”.

82. The method of embodiment 72, wherein said immunomodulator comprisesIL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-γ, TNF-αor a combination thereof.

83. The method of embodiment 72, wherein said radionuclide has an energybetween 20 and 10,000 keV.

84. The method of embodiment 83, wherein said radionuclide is selectedfrom the group consisting of ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁸Y, ²²⁵Ac, ¹⁷⁷Lu, ¹⁸⁸Re,¹⁸⁶Re, and combinations thereof.

85. The method of embodiment 72, wherein said photoactive therapeuticagent is a chromogen or dye.

86. The method of embodiment 77, wherein said alkylating agent isdacarbazine.

87. The method of embodiment 86, wherein said MN-14 antibody or fragmentthereof is administered in a dosage of 100 to 600 milligrams protein perdose per injection.

88. The method of embodiment 87, wherein said MN-14 antibody or fragmentthereof is administered in a dosage of 300-400 milligrams protein perdose per injection.

89. A method for treating cancer comprising administering to a subject,either concurrently or sequentially, a therapeutically effective amountof an anti-CEA antibody or fragment thereof and at least one therapeuticagent, and optionally formulated in a pharmaceutically acceptablevehicle.

90. The method of embodiment 89, wherein the therapeutic agent isCPT-11.

91. The method of embodiment 90, wherein the anti-CEA antibody orfragment is administered prior to administration of CPT-11.

92. The method of embodiment 91, wherein the anti-CEA antibody orfragment is administered around 3 days prior to administration ofCPT-11.

93. The method of embodiment 89, wherein the therapeutic agent is DTIC.

94. The method of embodiment 89, wherein the therapeutic agent isoxaliplatin.

95. The method of embodiment 89, wherein the therapeutic agent is5-fluorouracil/leucovorin.

96. In a method of treating cancer with a non-antibody therapeuticagent, the improvement comprising pre-treating a subject suffering fromcancer with an anti-CEA antibody or a fragment thereof prior toadministration of the non-antibody therapeutic agent.

97. The method of embodiment 96, wherein the anti-CEA antibody ishMN-14.

98. The method of embodiment 96, wherein the therapeutic agent isCPT-11.

99. A method of treating cancer with an antibody comprisingadministering to a subject suffering from cancer, prior toadministration of the antibody, an agent that activates granulocytesand/or NK cells in order to increase effector function of the antibody.

100. The method of embodiment 99, wherein the agent is GM-CSF.

101. The method of embodiment 99, wherein the antibody is an anti-CEAantibody.

102. The method of embodiment 101, wherein the antibody is hMN-14.

103. A method of treating cancer with an anti-CEA antibody or fragment,comprising administering to a subject suffering from cancer, prior toadministration of the anti-CEA antibody or fragment, an amount ofinterferon effective to up regulate CEA expression in tumor cells.

104. The method of embodiment 103, wherein the anti-CEA antibody ishMN-14.

105. An antibody fusion protein comprising at least one CEA binding siteand at least one other binding site for the same or different antigen.

106. The antibody fusion protein according to embodiment 105, whereinthe CEA binding site binds to the same site as an MN-14 antibody.

107. The antibody fusion protein according to embodiment 105, which isbivalent and trivalent.

108. The antibody fusion protein according to embodiment 105, whereinone arm of the fusion protein is a Class III, anti-CEA MAb that targetsCD66e and another arm of the fusion protein is from another CEAcrossreactive antibody that targets CD66a-d.

109. The antibody fusion protein according to embodiment 105, whereinthe binding arms are scFv or Fab regions.

110. An antibody fusion protein according to embodiment 105, which is abispecific, trivalent protein comprising one arm reactive with CD66a-dand two arms reactive with only CEA (CD66e).

111. An antibody fusion protein according to embodiment 105, which is abispecific protein comprising two arms that bind to NCA50/90.

112. An antibody fusion protein according to embodiment 105, which is adiabody comprising one arm that binds to NCA50/90 and a second arm thatbinds to a Class III epitope of CEA.

113. An antibody fusion protein according to embodiment 112, wherein theNCA-50/90 arm is obtained from an hMN-3 antibody and the second arm thatbinds to a Class III epitope of CEA is obtained from hMN-14.

114. An antibody fusion protein according to embodiment 113, wherein thefusion protein lacks an Fc-domain to prevent activation of cytokinerelease from granulocytes or which has an Fc-domain that has beenmodified to prevent complement fixation and ADCC.

115. An antibody fusion protein according to embodiment 104, which is atriabody comprising one hMN-3 arm and two hMN14 arms. 115a. An antibodyfusion protein according to embodiment 104, which is a triabodycomprising one hMN-15 arm and two hMN14 arms.

116. An antibody fusion protein according to embodiment 104, comprisingat least one hMN-14 arm and at least one NP-3 arm.

117. An antibody fusion protein according to embodiment 116, whichcomprises an Fc-domain to enable complement fixation and activation ofADCC.

118. An antibody fusion protein according to embodiment 104, furthercomprising a therapeutic agent.

119. An antibody fusion protein according to embodiment 118, wherein thetherapeutic agent is a cytokine.

120. An antibody fusion protein according to embodiment 119, wherein thecytokine is interferon, a colony-stimulating factor, or an interleukin.

121. An antibody fusion protein according to embodiment 120, wherein thecolony-stimulating factor is GM-CSF or G-CSF.

The invention is further illustrated by, though in no way limited to,the following examples.

Example 1 Materials and Methods Monoclonal Antibodies and Cell Lines

TT, a human medullary thyroid cell line, was purchased from the AmericanType Culture Collection. The cells were grown as monolayers in DMEM(Life Technologies, Gaithersburg, Md.) supplemented with 10% fetalbovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml), andL-glutamine (2 mM). The cells were routinely passaged after detachmentwith trypsin, 0.2% EDTA.

MN-14 is a Class III anti-CEA MAb, reacting with CEA and unreactive withthe normal cross reactive antigen, NCA, and meconium antigen (Hansen etal., Cancer, 71:3478 (1993)). The construction and characterization ofthe humanized forms of MN-14 and LL2, the anti-CD22 MAb used here as anegative control, have been previously described. (Sharkey et al.,Cancer Res., 55:5935s (1995); Leung et al., Mol. Immunol., 32:1416(1995)). P3x63Ag8 (MOPC-21) is an irrelevant mouse myeloma IgG₁ obtainedfrom the American Type Culture collection (Rockville, Md.). Theantibodies were purified by protein A chromatography.

In Vivo Studies

Tumors were propagated in female nu/nu mice (Taconic Farms, Germantown,N.Y.) at 6-8 weeks of age by s.c. injection of 2×10⁸ washed TT cells,which had been propagated in tissue culture. Antibodies were injectedi.v., via the lateral tail vein, into the tumor-bearing animals. Detailson the quantities of antibodies injected and the time of administrationare indicated in the Results section for each study. Results are givenas tumor volumes of individual animals as well as the mean±SE. Tumorsize was monitored by weekly measurements of the length, width, anddepth of the tumor using a caliper. Tumor volume was calculated as theproduct of the three measurements. Statistical comparisons were madeusing the Student's T-test to compare tumor volumes and area under thegrowth curves.

Example 2 Combination Therapy of Naked hMN-14 and DTIC Delivered 2 Daysafter Injection of TT (Human Medullary Thyroid) Tumor Cells

In a previous study, naked hMN-14 and dacarbazine (DTIC) were given incombination to TT 2 days after tumor implantation, using 100 μg and 25μg doses of DTIC (days 2, 3, and 4) and 250 μg doses of hMN-14 given onday 2, then weekly. The 100 μg DTIC dose combined with hMN-14 was moreeffective than either treatment alone (FIG. 1A). However, the 100 μgDTIC dose yielded too strong a response, while the 25 μg dose was noteffective. Surprisingly, the effects of MN-14 alone and DTIC alone werenot additive. In other words, given the results of treatment with 250 μghMN-14 alone and 100 μg DTIC alone, one would not predict that thecombination of 250 μg hMN-14 and 100 μg DTIC would have such apronounced effect. See FIG. 1A.

In this study, treatment began 2 days after TT cell injection, as in theprevious study. hMN-14 was given at 100 μg/dose on days 2, 3, 4, 5, 7,8, 9, 10, 11, 15 and 22, then every 7 days until the animal died, thetumor attained a volume of 2.0 cm³ or the study terminated for humanereasons. Doses of DTIC were 50 and 75 μg per dose, which is between thedoses given in the previous study. TT cells were injected subcutaneouslyin 60 nude mice. The day of injection was Monday, day 0, See FIG. 1B.

Results demonstrate that significant delays in tumor growth were causedby either MAb therapy alone or chemotherapy alone (FIG. 1B). The 75 μgdose of DTIC in combination with this schedule of hMN-14 antibody wassignificantly more effective than either treatment alone (p<0.02).Unexpectedly, the results of combined DTIC and MAb therapy were notadditive. At 7 weeks, 8/10 mice in the 75 μg DTIC and MAb group had nopalpable tumor, compared to 1/10 mice in the 75 μg DTIC only group and0/10 mice in the untreated and MAb group.

Mean tumor volumes at 7 weeks were 0.018±0.039 cm³ (75 μg DTIC plusMN-14), 0.284+0.197 cm³ (75 μg DTIC only), 0.899±0.545 cm³ (hMN-14 only)and 1.578±0.959 cm³ (untreated). Combined therapy of the naked anti-CEAantibody with DTIC augments the anti-tumor effects of antibody orchemotherapy alone, without increased toxicity. The superiority of thecombined modality treatment was surprising.

Dosing Summary: (1) hMN-14 was given daily (i.p.), except Sundays, at100 μg/dose/mouse on days 2 through 11. The antibody treatment wasinitiated on the same day as DTIC treatment. (2) DTIC was given on days2, 3, and 4 at 50 and 75 μg/dose, which corresponded to 5% and 7.5% ofthe MTD. Only one course of DTIC was given.

Groups: 6 groups of mice, each group containing 10 mice.

Group 1: Untreated.

Group 2. DTIC at 50 μg/dose, days 2, 3, and 4 (Wednesday, Thursday, andFriday).

Group 3. DTIC at 75 μg/dose, days 2, 3, and 4.

Group 4. DTIC at 50 μg/dose, days 2, 3, and 4, plus hMN-14 (100 μg/dose)day 2, 3, 4, 5, 7, 8, 9, 10, 11, 15 and 22, then every 7 days until theanimal died, the tumor attained a volume of 2.0 cm³, or the studyterminated.

Group 5. DTIC at 25 μg/dose, days 2, 3, and 4, plus hMN-14 (100 μg/dose)day 2, 3, 4, 5, 7, 8, 9, 10, 11, 15 and 22, then every 7 days until theanimal died, the tumor attained a volume of 2.0 cm³, or the studyterminated.

Group 6. hMN-14 (confirm 100 μg/dose), days 2, 3, 4, 5, 7, 8, 9, 10, 11,15 and 22, then every 7 days until the animal died, the tumor attained avolume of 2.0 cm³, or the study terminated.

Animals were monitored for survival. Tumor and body weight were measuredweekly.

Protocol: On day 2, 200 mg/vial DTIC was reconstituted with 19.7 mlsterile water for injection. The resulting solution contained 10 mg/mlof dacarbazine with a pH range of 3.0-4.0. The solution was used asneeded for the dilutions described below and the remainder was frozen in1 ml aliquots for subsequent use.

Groups 2 and 4: 5 ml of 0.5 mg/ml solution was prepared. 100 μl of 0.5mg/ml/mouse was injected i.v.

Groups 3 and 5: 5 ml of 0.75 mg/ml solution was prepared. 100 μl of 0.75mg/ml/mouse was injected i.v.

Quantity of hMN-14 was estimated. 100 μl of 1 mg/ml hMN-14 was injectedi.p. in mice in Groups 4, 5 and 6.

Example 3 Radioimmunotherapy Studies in a Human MTC Xenograft Model

Applicants developed a model for experimental radioimmunotherapy of MTCwith radiolabeled anti-CEA MAbs using human MTC xenografts of the CEA-and calcitonin producing human MTC cell line designated TT ([Stein, 1999#82], see Appendix). MTC tumors were established in nude mice by a s.c.inoculation of 2×10⁸ cells and allowed to grow for 2-5 weeks beforeinjection of MAbs. Biodistribution and RAIT studies were then carriedout with MN-1 4, which was shown by flow cytometry to react with TTcells. Both Ag8 and Mu-9 were used as negative control MAbs in thesestudies. Preliminary studies using smaller tumors of ˜0.08 g showed that7 days after the injection of ¹³¹I-MN-14, the percent of injected doseper gram of tumor (% ID/g) was 68.9% compared with only 12.6% ID/g forthe co-injected ¹²⁵I-Ag8 control. Using larger tumors, (grown for fiveweeks in nude mice; mean tumor weight=0.404 g), the % ID/g of tumorobserved at seven days post injection of ¹²⁵I-MN-14 was 12.4%. However,the % ID/g of the co-injected ⁸⁸Y-MN-14 was 50.5%, or 4.1-fold higherthan ¹²⁵I-MN-14. The tumor-to-blood, lungs, liver, spleen, and kidneyswere also higher with ⁸⁸Y-MN-14 than with ¹²⁵I-MN-14, while thetumor-to-bone ratios were equal with both agents. When ¹²⁵I-MN-14 and⁸⁸Y-MN-14 biodistribution data were used to predict the tumor dosimetrywith ¹³¹I-MN-14 and ⁹⁰Y-MN-14, respectively, the radiation absorbed dosedelivered at the MTD of ⁹⁰Y-MN-14 (115 μCi) was 1.75-fold higher thanthat delivered at the MTD of ¹³¹I-MN-14 (275 μCi) (4900 cGy vs. 2800cGy).

Therapy studies in this model confirmed that ⁹⁰Y-MN-14 is a bettertherapeutic agent than ¹³¹I-MN-14. In 5-week-old tumors, a 5-weekcomplete inhibition of tumor growth was seen at the MTD of ⁹⁰Y-MN 14compared to only a tumor growth delay with ¹³¹I-MN-14 (FIG. 2).Moreover, when smaller 2-week old tumors were treated, an average of 60%tumor volume reduction, with some complete tumor regressions, was seenat the MTD of ⁹⁰Y-MN-14. These anti-tumor effects were very significantcompared with the relatively rapid tumor growth in untreated animals orthose treated at the MTD of control MAbs. Thus, our preclinical studiesdemonstrated that this animal model is exquisitely suitable forexperimental RAIT with anti-CEA MAbs.

The longer path length and higher energy of ⁹⁰Y compared with ¹³¹I, inaddition to the fact that ⁹⁰Y is retained longer by target cells led todelivery of an increased radiation dose to tumor and thus more effectivetherapy at equitoxic doses. If our results with residualizing ¹³¹I(refs) can be generalized to MN-14 in MTC, we would expect thatresidualizing ¹³¹I would be at least equally effective to ⁹⁰Y in tumorsof the size studied here, and most likely superior in the setting ofmicrometastatic disease or as adjuvant therapy following surgery.

Example 4 Chemotherapy

Four drugs, doxorubicin, DTIC (dacarbazine), cyclophosphamide, andvincristine, were evaluated, singly and in combination, for their effecton the growth of TT MTC xenografts in nude mice. Doses were selectedbased on the doses of each drug given clinically to humans on a mg/m²basis. Animals were monitored for survival, and tumor volumes and bodyweights were measured weekly. FIG. 3 shows the tumor growth curve foranimals in this study. Given individually, doxorubicin, DTIC andcyclophosphamide, but not vincristine, yielded significant growthinhibition, although the growth delay caused by DTIC was markedly longerthan that of the other drugs. Approximate mean time to doubling for eachgroup was: untreated, 1 week; doxorubicin, 2.5 weeks; DTIC, 7.5 weeks;cyclophosphamide, 3 weeks; and vincristine, 1.5 weeks. Combiningdoxorubicin and DTIC improved the efficacy compared to either drugalone, increasing the mean time to doubling to 10 weeks. However, theincreased efficacy of doxorubicin and DTIC combination did not reach the95% confidence level in comparison to DTIC alone. The P values for AUCcomparisons were as follows: P<0.01 for doxorubicin+DTIC versusdoxorubicin, and P<0.1 for doxorubicin+DTIC versus DTIC. The 4-drugregimen extended the mean time to doubling to 12 weeks; P<0.01 forcomparisons to both doxorubicin and DTIC.

Log rank analysis of survival data for the individual drugs versus theuntreated group indicated a significant difference only for DTIC andcyclophosphamide. Mean survival time for the untreated control group was4 weeks compared to 11 weeks and 8 weeks for DTIC and cyclophosphamidetreatment groups, respectively, and greater than 12 weeks for the drugcombinations. Toxicity, as measured by body weight loss, was within theacceptable range for all study groups. Maximum weight loss was observed1 week after treatment in the mice treated with all 4 drugs, rangingfrom 3-12% loss of body weight.

Example 5 Combining Radioimmunotherapy and Chemotherapy for Treatment ofMTC

RAIT Plus 4-Drug Combination.

The effect of combining RAIT with ⁹⁰Y-anti CEA MAb MN-14 and the 4-drugcombination was evaluated by comparing the growth of TT in untreatedmice to those treated with the 4-drug regimen described above(doxorubicin, DTIC, cyclophosphamide, and vincristine), 100% of themaximum tolerated dose (MTD) of RAIT (105 μCi), 50% of the MTD of RAIT,and 50% of the MTD of RAIT combined with the 4 drugs. FIG. 4 shows thegrowth curves of TT tumors in mice given the various treatment regimens.All four of the treatment groups yielded significant improvement inefficacy compared to the untreated animals. Whereas the approximate meantime to doubling in the untreated animals was 1.5 weeks, chemotherapywith the 4 drugs extended the mean doubling time to 10 weeks and RAITalone yielded 4-week and 8-week doubling times at 50% and 100% of theMTD, respectively. As expected, both the 100% RAIT group and the 4-drugtherapy regimen were significantly better than the 50% RAIT group. Mostimportantly, combining 50% RAIT and the 4-drug regimen yielded improvedresults, compared to either therapy alone, further extending the meandoubling time to approximately 12.5 weeks. For the comparison of thecombined treatment to the 4-drug regimen, P<0.02, and for the comparisonto 100% RAIT, P<0.01.

Mean weight loss 1 week post treatment (nadir) was 9% for the 100% RAITand the 4-drug regimens, but 15% for combined 50% RAIT plus 4-drugtreatment. In addition, in the combined therapy group, one animal diedthree weeks post treatment and a second animal had a weight loss greaterthan 20%. Thus, this treatment exceeded the maximum tolerated dose.

RAIT Plus Chemotherapy with 2-Drug Regimens.

The effect of combining RAIT with ⁹⁰Y-anti CEA MAb MN-14 andchemotherapy with a 2-drug combination, consisting of doxorubicin andDTIC, was also evaluated in this MTC xenograft model. Approximatedoubling times for the groups were: untreated, 1.5 weeks; doxorubicinplus DTIC, 8 weeks; the MTD of RAIT, 10 weeks; and the MTD of RAITcombined with 25-75% of the 2-drug regimen, greater than 12 weeks. Thus,RAIT alone was more effective than the 2-drug regimen and, mostsignificantly, combining RAIT and the 2-drug regimen yielded improvedresults compared to either therapy alone. For the comparison of thecombined treatment to the 2-drug regimen, P<0.005, for the comparison toRAIT alone, P<0.02.

Mean weight loss 1-2 weeks post treatment (nadir) was 2-8% for allgroups, except the 100% RAIT plus 75% 2-drug chemotherapy group, where a13% loss was observed at 2 weeks. In addition, in this combined therapygroup, two animals died 3-4 weeks post treatment and one experienced aweight loss greater than 20%. Thus, addition of the 75% dose level ofdoxorubicin and DTIC to 100% RAIT treatment exceeded the MTD, whereas50% of this 2-drug combination can be tolerated in combination with 100%RAIT.

RAIT Plus Doxorubicin.

Because previous publications have reported the combination of RAIT withdoxorubicin in this model (Stein et al., Clin Cancer Res., 5:3199s(1999); Behr et al., Cancer Res. 57:5309 (1997)), a direct comparisonwas made to the RAIT plus doxorubicin regimen. A direct comparison wasalso made to RAIT plus the 4-drug regimen. All treatments yieldedsignificant efficacy compared to the untreated animals. The meandoubling time for the RAIT plus doxorubicin group was 12 weeks. In thisstudy combining the full MTD of RAIT with either 50% of doxorubicin andDTIC or the 4-drug regimen extended the mean doubling time to greaterthan 15 weeks, with no statistically significant difference betweenthese two groups. A substantial number of objective responses wereobserved in these studies. Following treatment with RAIT plusdoxorubicin there were 3 complete responses, 2 partial responses, and 5animals with stable disease for at least 4 weeks, out of a total of 10mice. The RAIT plus 2-drug protocol increased the objective responses to10 complete responses and 2 partial responses of 12 animals, and theRAIT plus 4-drug treatment protocol led to 7 complete responses and 2partial responses out of 9 mice.

RAIT Plus DTIC.

Because DTIC was the most effective chemotherapeutic agent whenadministered alone, the efficacy of RAIT plus DTIC was evaluated incomparison to that of RAIT plus doxorubicin and DTIC. Omittingdoxorubicin from the treatment protocol will be important for clinicalapplication in order to avoid the added toxicity of this drug,especially the known cardiac toxicity. As shown in FIG. 5, the two studygroups which received the chemotherapy in combination with RAIT, eitherdoxorubicin and DTIC or DTIC only, are approximately equal to eachother, and both are more effective than the single modality treatments.P values for AUC comparisons were as follows: P<0.01 for RAIT+DTICversus DTIC, and P<0.05 for RAIT+DTIC versus RAIT. The mean doublingtime for the RAIT plus DTIC, and RAIT plus doxorubicin and DTIC groupswere 15.5 weeks and 14 weeks, respectively, compared to 7.5 weeks and 9weeks for DTIC and RAIT alone, respectively. Thus, the combined modalitytreatment of RAIT plus DTIC extended the mean time to doubling by 100%over the DTIC chemotherapy. No significant difference was observed byeither AUC or log rank analyses between the RAIT plus DTIC, and RAITplus doxorubicin and DTIC groups.

Example 6 Studies with Naked Anti-CEA Alone

Therapy with Naked hMN-14

To study the effect of unlabeled hMN-14 on the growth of TT tumors innude mice, a single injection of hMN-14 was administered i.v. either oneday or eleven days post tumor cell injection. FIG. 6 shows the tumorgrowth curves of animals treated with 0.5 mg hMN-14/mouse compared tountreated controls. The untreated group contained 16 animals; the twotreatment groups contained 10 animals each. A significant growth delaywas observed between the untreated group and the group treated on day-1post tumor injection. Significant differences in the mean tumor sizes(p<0.05) were observed from day-32 through day-93. Between day-32 andday-60 there was a 64-70% inhibition of tumor size in the MN-14 treatedgroup compared to the untreated animals. There were no significantdifferences between the mean tumor sizes in the day-11 group anduntreated animals. Significant delay in tumor growth was also seen byt-test analysis of the area under the growth curves. P<0.05 for theuntreated group compared to the group treated one day following tumorinjection, but not for the group treated eleven days following tumorinjection.

Specificity of Treatment

FIG. 7 summarizes the results of a study on the specificity of theanti-tumor response. The effect of unlabeled hMN-14 on the growth of TTtumors in nude mice was compared to that of a negative control humanizedMAb, hLL2 (anti-CD22), and the murine MN-14. MAbs (0.5 mg/mouse) wereadministered (i.v.) one day after TT cells, then three additional weeklydoses of 0.5 mg/mouse were given. Groups of 15 animals were studied. Thegrowth inhibition observed in the first study from treatment with 0.5 mghMN-14 was confirmed in this study. Significant differences in meantumor sizes (p<0.05) between the hMN-14 and the untreated group wereobserved starting at day-23. At day-37 the mean tumor volume in thegroup treated with hMN-14 was 42.7% of the untreated control animals.Treatment with murine MN-14 yielded results similar to the hMN-14.Treatment with hLL2 did not slow tumor growth; instead there was a small(not significant) increase in growth rate. For example, at day-37 87% ofthe tumors treated with hMN-14 were less than 0.5 cm³, compared to 40%of the untreated and 29% of the hLL2 treated group. T-test analysis ofthe area under the growth curves demonstrated significant differences(p<0.05) between the untreated group and the groups treated with eitherhMN-14 or murine MN-14, but not the group treated with hLL2. Inaddition, the hMN-14 group was significantly different from the hLL2group but not the murine MN-14 treated animals.

Effect of Dose

To study the effect of dose of unlabeled hMN-14 on the growth of TTtumors in nude mice, increasing doses of hMN-14 were evaluated. Antibodydoses were administered 1 day after TT cells, then weekly until thetermination of the study. Weekly doses ranged from 0.125 mg to 2.0 mghMN-14/mouse in groups of six mice. Significant differences in meantumor sizes and area under the growth curves between the untreated groupand all treatment groups were observed (FIG. 8). For example, betweenday-21 and day-49 mean tumor volume in the 2 lowest hMN-14 treatmentgroups were 27-40% of the size of tumors in the untreated animals.Treatment with the lower doses, 0.125 mg and 0.25 mg, appeared to bemore effective than treatment with the higher doses, although thedifference did not reach statistical significance.

Timing

The effect of time between TT injection and initial dose of hMN-14 onthe growth of TT tumors in nude mice was evaluated by varying the day ofadministration of MAb. hMN-14 (0.25 mg) was administered either 1, 3, or7 days after TT cells, then weekly until termination of the study.Groups of 7-8 animals were studied. Results are summarized in FIG. 9.Significant differences in mean tumor sizes (p<0.05) between theuntreated group and all three treatment groups were observed. However,the difference in mean tumor size between the untreated mice and theday-7 treatment group was only significant at one time point, day-28.Day-1 treated mice yielded significant differences from 21-77 days, andday-3 treated mice yielded significant differences from 21-70 days.T-test analysis of the area under the growth curves indicatedsignificant growth inhibition for the groups treated with hMN-14 either1 or 3 days after TT cell administration compared to untreated group.This analysis did not reach the 95% confidence limit for differencebetween the untreated group and the group treated on day-7 (p=0.057 at 5weeks).

Example 7 Combined Naked Anti-CEA Plus DTIC Therapy of MTC

To study whether naked hMN-14 can add to the efficacy of DTIC, TTbearing nude mice were given DTIC (75 μg/dose) in combination with acourse of treatment of the unlabeled MAb. DTIC was administered for 3consecutive days at 75 μg/dose as one course, beginning 2 days afters.c. injection of TT cells. hMN-14 MAb treatment was initiated on thesame day as the first dose of DTIC, at 100 μg/dose/day for 5 days in thefirst two weeks, then twice weekly. Significant delays in tumor growthwere caused by these schedules of either MAb therapy or chemotherapyalone (FIG. 10). The 75 μg dose of DTIC in combination with thisschedule of hMN-14 was significantly more effective than eithertreatment alone (P<0.02). At 7 weeks, 8/10 mice in the 75 μg DTIC+MAbgroup had no palpable tumor, compared to 1/10 in the 75 μg DTIC-onlygroup and 0/10 in the untreated and MAb-only groups. Mean tumor volumesat 7 weeks were 0.018+0.039 cm³ (75 μg DTIC+hMN-14), 0.284+0.197 cm³ (75μg DTIC), 0.899+0.545 cm³ (hMN-14) and 1.578+0.959 cm³ (untreated).

The anti-CEA MAb MN-14 has shown unexpected anti-tumor efficacy in MTCwithout conjugation to a cytotoxic agent. Differences in mean tumorsizes between the hMN-14 treated and the untreated groups were observedbeginning at 3 weeks and lasting at least 2 months. Treatment withisotype matched negative control MAbs did not slow tumor growth. This isthe first evidence of tumor suppression with a “naked” anti-CEA MAb.However, combined therapy of the naked anti-CEA MAb with DTIC augmentsthe anti-tumor effects of antibody or chemotherapy alone, withoutincreased toxicity. The superiority of the combined modality treatmentargues for the integration of CEA-MAb therapy into chemotherapeuticregimens for MTC management.

Example 8

FIG. 15 shows the effects of naked hMN-14 CEA MAb and DTIC treatment ina medullary thyroid cancer model. Treatment was initiated 2-days aftertumor transplantation. DTIC was administered at −75 μg on days 2, 3, and4 at 7.5% of the MTD to mice. hMN-14 was administered at 100 μg/day ondays 2-5, 7-10, 11, 15, 22, and then once per week. The results show astatistically significant difference (P<0.05) between the areas underthe curve for all groups. Naked hMN-14 CEA MAb treatment showed asignificant effect on inhibiting tumor growth. When combined with DTIC,a surprisingly enhanced level of inhibition of tumor growth occurredrelative to either treatment alone.

Example 9 Naked Anti-CEA Antibody Treatment Plus CPT-11 or 5-FU in ColonCancer Cells

The present experiment discloses the in vitro and in vivo effect of ahumanized, naked anti-CEA, hMN-14 antibody (hMN-14) alone, and incombination with chemotherapy on colon cancer growth.

Methods and Materials

Antibody Production. The CDR-grafted (humanized) MN-14 (hMN-14)anti-carcinoembryonic antigen (CEA) (Sharkey, R. M., et al., Cancer Res,55: 5935-5945, 1995) along with the murine MN-14 and other antibodiestargeting different CEA epitopes (NP1, NP3, MN3, MN15; (Sharkey, R. M.,et al., Cancer Res, 50: 2823-2831, 1990) were purified by protein A andion-exchange chromatography (Q-Sepharose; Pharmacia, Piscataway, N.J.).Purity was tested by immunoelectrophoresis, polyacrylamide gelelectrophoresis using reducing and non-reducing conditions andsize-exclusion high-pressure liquid chromatography.

In Vivo Therapy Studies. Survival therapy studies were performed using aCEA-positive GW-39 intrapulmonary micrometastasis model (Sharkey, R. M.,et al., J Natl Cancer Inst., 83: 627-632, 1991; Blumenthal, R. D., etal., Cancer Res, 52: 6036-6044, 1992). Stock subcutaneous GW-39 humancolorectal tumors were used to prepare a 10% or 5% cell suspension.Cells (30 μl) were injected i.v. into the caudal vein. HuMN-14 IgG wasinitiated on either day 0 or day 3 after cell implantation andadministered daily×14 days and twice weekly thereafter for the durationof the study at a dose of 100 μg/d. CPT-11 was administered at a dose of160 μg daily for 5 days i.p. (20% of the MTD) starting on day 0 or day 3after cell implantation. For some studies, the stock GW-39 tumor camefrom mice that received 100,000 U of IFNγ twice daily for 4 days toupregulate CEA expression (Greiner, J. W., et al., 16: 2129-2133, 1996),which was confirmed by immunohistology as previously described(Blumenthal, R. D., et al., Int. J. Cancer, 51: 935-941, 1992). Bodyweight was monitored weekly and animal survival recorded. Results wereanalyzed with the Kaplan-Meir test and median survival time determined.

In Vivo Effects of Antibody-Induced Chemosensitization of Cancer Cells.

The effect of hMN14-induced chemosensitization was apparent in vivo aswell as in vitro. Survival curves for mice bearing GW-39 intrapulmonarymicrometastases, as described above, and untreated or treated with hMN14alone (100 μg/d×14 d and twice weekly for the duration of the study), a10% MTD of CPT-11 (80 μg/d×5 days) alone or both modalities together.Treatment was initiated the day of cell implantation (30 μl of a 10%GW-39 cell suspension). Each treatment group started with 10 mice andthe study was repeated twice. The results show that co-administration ofhMN-14 and CPT-11 to nude mice bearing GW-39 lung micrometastasesincreases survival beyond the effect of either modality alone.Administration of a 10% MTD of CPT-11 resulted in a 1-week increase inmedian survival from 56 days to 63 days (p<0.05). Median survival timeof animals dosed with both hMN-14 and CPT-11 on day 0 increased by anadditional 2 weeks to 77 days (p<0.005 compared with untreated mice).Since maximal antibody accretion occurs 3 days post injection, hMN-14treatments were initiated 3-days before CPT-11 to determine whether suchdosing would further enhance the therapeutic effect of the combinedmodality treatment approach by allowing high antibody uptake andchemosensitization in vivo. The results demonstrate that the 3-daypretreatment with hMN-14 followed by CPT-11 increased median survival to105 days (p<0.001), compared with CPT-11 alone on day 3, with a mediansurvival of 70 days. In this study, co-treatment of hMN-14 and CPT-11was superior, as evidenced by a median survival of 70 days vs. CPT-11alone on day 0, with a median survival of 63 days or untreated mice witha median survival of 35 days. The results were similar for a furtherexperiment where a 5% GW-39 cell suspension was used instead of the 10%GW-39 cell suspension.

Example 10 In Vivo Effect of Pretreatment with an Immunomodulator Priorto Treatment with hMN-14 and CPT-11 on Tumor Cell Chemosensitivity

A further experiment evaluated the combined treatment of hMN-14 withCPT-11, initiated together in mice with GW-39 tumors expressing higherCEA levels, as a result of pretreatment of GW-39 stock tumors (10% GW-39cell suspension) with interferon-γ (IFNγ). The experiments involvinginterferon-gamma enhancing the antitumor effects of naked CEA antibody(hMN-14) were conducted as follows.

First, GW-39 human colon cancer was grown subcutaneously in a mouse thatreceived 100,000 units of IFN-gamma twice a day for 4 days. A controlmouse with GW-39 tumor was not given IFN. Experimental mice wereinjected i.v. with a 5% suspension of GW-39 (w/v) from either of the twomice (i.e., with or without IFN treatment) into two groups of eight.Four of each received tumor from the IFN-treated mice and four from theuntreated mice. One group of 8 mice then received hMN-14 (100 ug perday×14 days and then twice weekly thereafter until expt was ended),another group CPT-11 at 160 ug/day×5 days (=20% of maximum tolerateddose), a third group received the same doses of antibody+drug combined,and a fourth group that was not treated at all. Animal weights weremeasured and survival determined weekly. Also, samples of stock tumortreated with IFN in the mice that were later implanted were alsoprocessed for immunohistology to assess increase in CEA expression inthe tumors from mice treated with IFN-gamma, and this was controlled byalso treating the suspensions by immunohistology with an irrelevant IgG,such as Ag8, which showed no CEA staining.

Example 11

A comparison was performed of the effects of naked hMN-14 CEA Mab on lowand high (induced by interferon-gamma, as described earlier)CEA-expressing tumor cells in an animal model. The results demonstratethat increased expression of CEA antigen on tumor cells correlates withimproved efficacy of anti-CEA antibody. The results of the comparisonstudy are shown in FIG. 21. Thus, interferon-gamma pre-treatment isuseful to boost the efficacy of anti-CEA antibody therapy in thetreatment of cancer.

Example 12 Sigmoid Colon Cancer Therapy with CEA Antibody and GM-CSF

J. R. is a 62-year-old man who is refractive to chemotherapy with5-fluorouracil and leukovorin to reduce his metastases to the liverfound at the time of discovery and removal of his sigmoid colon cancer.His plasma titer of carcinoembryonic antigen (CEA) at presentation is 34ng/mL, and computed tomography of the liver shows several small lesionsmeasuring between 2 and 4 cm in diameter in the right lobe; otherradiological studies appear to be normal. Immunotherapy with humanizedanti-CEA IgG_(I), hMN-14, monoclonal antibody is begun on a weekly basisfor 4 weeks, at an intravenous dose of 300 mg/m²) infused over 2 hours.One week prior to hMN-14 therapy, the patient receives 2 subcutaneousinjections of 200 mcg/m² GM-CSF (sargamostim, Leukine®), 3 days apart,and continued twice weekly during the 4 weeks of hMN-14 therapy. Afterthese four weeks, both hMN-14 and GM-CSF are given at the same dosesevery other week for an additional 3 months, but the dose of GM-CSF isincreased to 250 mcg/m². Prior to each administration of the humanizedCEA antibody, the patient is given diphenhydramine (Benadryl®), 50 mgorally, and acetaminophen (Tylenol®), 500 mg orally. At this time, thepatient is restaged, with CT measurements made of the liver metastasesand diverse radiological scans of the rest of the body. Blood is alsotaken for chemistries and for determination of his blood CEA titer. Noareas of disease outside of the liver are noted, but the sum of thediameters of the measurable tumors in the liver appear to decrease by 40percent, and the patient's blood CEA titer decreases to 18 ng/mL, thusindicating a therapeutic response. Immunotherapy with hMN-14 and GM-CSF,given once every other week at 200 mg/m² for hMN-14 and 250 mcg/m² forGM-CSF, are administered for another 2 months, and restaging showsadditional decrease in the sum of the diameters of the liver tumors anda fall in the CEA titer to 10 ng/mL. Since tumor decrease is measured asbeing >65% over the pre-therapy baseline, the therapy is considered tohave provided a partial response. After this, the doses were made lessfrequent, once every month for the next six months, and all studiesindicate no change in disease. The patient is then followed for another10 months, and remains in a partial remission, with no adverse reactionsto the therapy, and generally without any symptoms of disease.

Example 13 Combined Immunotherapy and Chemotherapy of Metastatic ColonCancer

S. T. is a 52-year-old woman presenting with liver and lung metastasesof colon cancer following resection of the primary tumor. She is placedon a combined chemotherapy and immunotherapy protocol based on theGramont schedule (A. de Gramont et al., J Clin Oncol. 2000;18:2938-1947), but with the addition of humanized anti-CEA monoclonalantibody IgG₁. Prior to infusions of the antibody, she receives 50 mgorally of diphenhydramine (Benadryl®) and 500 mg orally of acetaminophen(Tylenol®). She receives a 2-hr infusion of leucovorin (200 mg/m²/day)followed by a bolus of 5-fluorouracil (400 mg/m²/day) and 22-hourcontinuous infusion of 5-fluorouracil (600 mg/m²/day) for 2 consecutivedays every 2 weeks, together with oxaliplatin at 85 mg/m² as a 2-hrinfusion in 250 mL of dextrose 5%, concurrent with leukocorin on day 1(FOLFOX4 schedule). The patient also receives anti-emetic prophylaxiswith a 5-hydroxyltryptamine-3-receptor antagonist. One week prior tothis 2-week chemotherapy cycle, hMN-14 monoclonal anti-CEA antibody isinfused over 2 hrs at a dose of 200 mg/m², and repeated each week of the2-week chemotherapy cycle, and every week thereafter for the next monthwith another chemotherapy cycle. Also, a subcutaneous dose of 5mcg/kg/day of G-CSF (filgrastim, Neupogen®) is administered once weeklybeginning with the second chemotherapy cycle, and continued at this dosefor the duration of immunotherapy with hMN-14 antibody, over the next 3months. A total of 5 cycles of chemotherapy with continuedadministration of hMN-14 antibody and filgrastim. Thereafter, hMN-14 andfilgrastim therapy is given, at the same doses, every other week for thenext 3 months, without chemotherapy. The patient is staged 2 monthslater, and her liver and lung metastases show shrinkage by computedtomography measurements of >80 percent of disease measured in the liverand lungs, as compared to the measurements made prior to therapy. Herblood CEA titer also shows a drop from the pre-therapy level of 63 ng/mLto 9 ng/mL. She is followed over the next 6 months, and her diseaseappears to be stable, with no new lesions found and no increase in thedisease remaining in the liver and lungs. The patient's predominanttoxicity is peripheral sensory neuropathy, which consists oflaryngeopharyngeal dysesthesia. The patient also experiences diarrhea,mucositis, nausea and vomiting during the chemotherapy cycles, but theseare not excessive. She does not experience any adverse events when onlyimmunotherapy is administered, and is able to return to full-timeactivities without any significant restrictions.

Example 14

FIG. 16 shows the effects of naked hMN-14 CEA Mab and CPT-11 treatmentin an advanced colon cancer model. hMN-14 was given to mice at a dose of100 μg/day over 14 days and then 2 times/week thereafter, starting onday 0 after tumor implantation. CPT-11 was given at 60 μg/day over 5days. No effect of hMN-14 by itself is apparent under these conditions,and only a modest effect of CPT-11 (p<0.05) was observed. However,hMN-14 increases the effect of CPT-11, as seen by comparing the CPT-11median survival of 63 days vs. combination therapy median survival of 77days (p<0.005). Combination therapy with hMN-14 CEA Mab and CPT-11significantly prolongs survival of an animal with advanced human colonictumor metastasis.

Example 15

FIG. 17 shows the effects of naked hMN-14 CEA MAb and CPT-11 treatmentin a low tumor burden cancer model. In a reduced tumor burden modelutilizing a 5% tumor cell suspension, CPT-11, hMN-14 alone, andcombination therapy of hMN-14+CPT-11 were compared. Dosages were asindicated in Example 14. There was no apparent effect of hMN-14 aloneunder these conditions. CPT 11 alone resulted in a median survival timeof 70 days. By contrast, the combination therapy produced a mediansurvival time of 91 days (p<0.025). The combination of hMN-14 and CPT-11significantly prolongs survival of animals with low tumor burden in ametastatic model of human colonic cancer.

Example 16

FIG. 18 shows the effects of pre-treatment with naked hMN-14 CEA Mabgiven 3 days prior to CPT-11 treatment in a cancer model. In a reducedtumor burden model utilizing a 5% tumor cell suspension, CPT-11, hMN-14alone, and combination therapy of hMN-14+CPT-11 where the hMN-14 wasadministered 3 days prior to the CPT-11 were compared. Dosages were asindicated in Example 14. hMN-14 alone increased median survival time by21% (p<0.05) under these conditions. CPT 11 alone increased survival by76% (p<0.001). By contrast, the combination therapy where hMN-14 isadministered 3 days prior to CPT-11 produced a median survival timeincrease of an additional 58% above CPT-11 alone (p<0.001 compared withCPT-11 alone). Pre-treatment with hMN-14 significantly prolongs survivalof animals with low tumor burden in a metastatic model of human coloniccancer.

Example 17

FIG. 19 shows a comparison of various administration schedules of nakedhMN-14 CEA MAb and CPT-11 in a human colon cancer model. Giving hMN-14 3days before CPT-11 is the most effective. Dosages were as indicatedExample 14. When the order is reversed (CPT-11 is given 3 days beforehMN-14) or when both are given together at the same time, mediansurvival time of 70 days was an increase over the untreated controlgroup (35 days) but was still significantly less than the mediansurvival time of 105 days with the hMN-14 pre-treatment 3 days beforeCPT-11.

Example 18

FIG. 20 shows the effects of GM-CSF pre-treatment on naked hMN-14 CEAMab therapy in a human colon cancer model. GM-CSF was administered at adose of 1 μg/mouse/day on days −4, −3, −2, and −1. Tumor cells wereimplanted at day 0 along with hMN-14 treatments. Other dosages were asindicated Example 14. The GM-CSF pre-treatment resulted in astatistically significant increase in median survival time (p<0.002)over either GM-CSF alone or hMN-14 alone.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention.

All of the publications and patent applications and patents cited inthis specification are herein incorporated in their entirety byreference.

1. A method for treating a CEA expressing cancer comprising: a)administering to a subject with a CEA expressing cancer a naked Class IIanti-CEA monoclonal antibody (MAb) or antigen-binding fragment thereof,wherein the Class II anti-CEA MAb is unreactive with human granulocytes;and b) administering either concurrently or sequentially at least oneanti-cancer therapeutic agent.
 2. The method of claim 1, wherein thetherapeutic agent is selected from the group consisting of a secondantibody or fragment thereof, an immunoconjugate, a cytotoxic agent, achemotherapeutic agent, a radionuclide, an immunomodulator, aphotoactive therapeutic agent, an antisense oligonucleotide and ahormone.
 3. The method of claim 2, wherein the therapeutic agentcomprises vincristine, doxorubicin, DTIC, cyclophosphamide, CPT-11,oxaliplatin, gemcitabine and 5-fluorouracil/leucovorin.
 4. The method ofclaim 2, wherein the therapeutic agent is selected from the groupconsisting of vincristine, doxorubicin, DTIC, cyclophosphamide, CPT-11,oxaliplatin, gemcitabine and 5-fluorouracil/leucovorin.
 5. The method ofclaim 2, wherein the therapeutic agent is a second naked antibody or animmunoconjugate.
 6. The method of claim 5, wherein the second nakedantibody is a Class III anti-CEA antibody.
 7. The method of claim 5,wherein the second naked antibody is a monovalent Class I anti-CEAantibody.
 8. The method of claim 5, wherein said second naked antibodyor immunoconjugate comprises a humanized, chimeric, human or murineantibody or fragment thereof reactive with an antigen selected from thegroup consisting of EGP-1, EGP-2, MUC-1, MUC-2, MUC-3, MUC-4, PAM-4,KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, a tumorangiogenesis antigen, Ga 733, tenascin and fibronectin.
 9. The method ofclaim 5, wherein the second naked antibody or immunoconjugate is amonovalent Class I anti-CEA antibody or fragment thereof or a Class IIIanti-CEA antibody or fragment thereof.
 10. The method of claim 1,wherein the cancer is medullary thyroid carcinoma or colon cancer. 11.The method of claim 2, wherein the cytotoxic agent is a drug or toxin.12. The method of claim 11, wherein the drug possesses a pharmaceuticalproperty selected from the group consisting of antimitotic, alkylating,antimetabolite, antiangiogenic, apoptotic, alkaloid, COX-2, andantibiotic agents and combinations thereof.
 13. The method of claim 11,wherein the drug is selected from the group consisting of nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, anthracyclines, taxanes, COX-2inhibitors, pyrimidine analogs, purine analogs, antimetabolites,antibiotics, enzymes, epipodophyllotoxins, platinum coordinationcomplexes, vinca alkaloids, substituted ureas, methyl hydrazinederivatives, adrenocortical suppressants, antagonists, endostatin,taxols, camptothecins, doxorubicins and a combination thereof.
 14. Themethod of claim 11, wherein the toxin is a microbial, plant or animaltoxin selected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin and Pseudomonas endotoxin.
 15. The method of claim 2, whereinthe immunomodulator is selected from the group consisting of a cytokine,a stem cell growth factor, a lymphotoxin, a hematopoietic factor, acolony stimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin, tumor necrosis factor (TNF), an interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-alpha,interferon-beta, interferon-gamma, a stem cell growth factor designated“S1 factor,” IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18 and IL-21. 16.The method of claim 2, wherein the immunomodulator comprises IL-1, IL-2,IL-3, IL-6, IL-10, IL-12, IL-18, interferon-γ, TNF-α or a combinationthereof.
 17. The method of claim 1, wherein combination of naked ClassII anti-CEA antibody and therapeutic agent is more effective for therapyof a CEA-expressing cancer than either antibody or therapeutic agentalone or the sum of the effects of naked anti-CEA antibody andtherapeutic agent.
 18. The method of claim 2, wherein the radionuclideis selected from the group consisting of ³²P, ³³P, ⁴⁷Sc, ⁵⁹Fe, ⁶⁴Cu,⁶⁷Cu, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁰Y, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ¹²⁵I, ¹³¹I,¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb, ²¹²Pb, ²¹³Bi, ⁵⁸Co, ⁶⁷Ga, ^(80m)Br,^(99m)Tc, ^(103m)Rh, ¹⁰⁹Pt, ¹¹¹In, ¹¹⁹Sb, ¹⁶¹Ho, ^(189m)Os, ¹⁹²Ir,¹⁵²Dy, ²¹¹At, ²¹²Bi, ²²³Ra, ²¹⁹Rn, ²¹⁵Po, ²¹¹Bi, ²²⁵Ac, ²²¹Fr, ²¹⁷At,²¹³Bi, ⁸⁸Y and ²⁵⁵Fm.
 19. The method of claim 1, wherein the subject hasa colon, rectal, pancreatic, breast, ovarian, thyroid or lung cancer.20. The method of claim 1, wherein said antibody fragment is selectedfrom the group consisting of a F(ab′)₂, a Fab′, a Fab, an Fv and anscFv.
 21. The method of claim 1, wherein the Class II anti-CEA antibodyis MN-6 or NP-3.